Live attenuated vaccines

ABSTRACT

The present invention refers to a method for the production of live attenuated bacterial strains, suitable as vaccine candidates, comprising the steps of: A. providing a bacterial strain capable of expressing glutamate racemase and possibly D-amino acid transaminase and comprising a peptidoglycan cell wail, and B. inactivating the gene or genes encoding for the glutamate racemase enzyme and, if needed, the gene or genes encoding for the enzyme D-amino acid transaminase in such way that the bacterial strain is no longer capable of expressing a functional glutamate racemase and/or a functional D-amino acid transaminase; wherein the inactivation of said genes causes said bacterial strain to be auxotrophic for D-glutamate.

FIELD OF THE INVENTION

Live attenuated bacteria vaccines are provided. Also provided aremethods by which such vaccines can be obtained.

BACKGROUND OF THE INVENTION

The means by which a warm blooded animal, including a human, overcomesmicrobial pathogenesis is a complex process. Immunity to microbialpathogenesis is one means by which a warm blooded animal avoidspathogenesis, or suffers a less intense pathogenic state. Incompleteimmunity to a given pathogen results in morbidity and mortality in apopulation exposed to a pathogen. It is generally agreed that vaccinesbased on live but attenuated micro-organisms (live attenuated vaccines)induce a highly effective type of immune response. Such vaccines havethe advantage that, once the animal host has been vaccinated, entry ofthe microbial pathogen into the host induces an accelerated recall ofearlier, cell-mediated or humoral immunity which is able to control thefurther growth of the organism before the infection can assumeclinically significant proportions. Vaccines based on a killed pathogen(killed vaccine) are generally conceded to be unable to achieve thistype of response. However, vaccines that contain a live pathogenpresent, depending on the level of attenuation, the danger that thevaccinated host upon vaccination may contract the disease against whichprotection is being sought. Therefore, it would be desirable to have avaccine that possesses the immunising attributes of a livemicro-organism but that is not capable of causing undesirable sideeffects upon vaccination.

However, it is important to note that the effective use of an attenuatedbacterial strain as a vaccine candidate cannot be predicted merely bysuch level of attenuation. In this regard, the general approach forattenuating bacteria is the removal of one or more virulence factors(genetic modified organisms—GMOs), in most cases, however, virulencefactors also play a role in inducing immunity as protective epitopes. Inthose cases, deletion of virulence factors unavoidably impairs theimmunogenic capacities of the bacterium. This is of course an unwantedsituation. Therefore, a live vaccine should preferably retain theantigenic complement of the wild type strain.

Moreover, once attenuation level is established, the immune response toa particular type of vaccine candidate and the success of a vaccinecomposition including such micro-organisms may still be influenced bymany factors as detailed below:

-   -   a. The live attenuated vaccine strain should preferably have        substantially no probability for reverting to its original state        (usually a virulent wild type strain) and none of the genes        manipulated should be complemented by other genes causing the        bacteria to be capable of causing disease (stable mutations are        preferred).    -   b. The presence of endotoxins in a live vaccine can be a        disadvantage if not considered as these molecules can cause        serious systemic reactions. Also, the administration of        whole-cell vaccines is a classical risk factor for local        reactogenicity (severe pain, local swelling and edema,        panniculitis or ulcer, etc).    -   c. The viability and fitness of the attenuated GMO should not be        drastically affected, as some replication is expected to occur        in the body to create enough of the micro-organism and its        antigens to stimulate the immune system. In fact, any mutation        in a gene may interfere with replication or may damage the live        micro-organism in the vial, causing the vaccine to be        ineffective. Therefore, each type of genetic modification must        be carefully evaluated for unexpected effects on the cell.    -   d. Moreover, gene sharing or protein moonlighting—a phenomenon        by which a protein can perform more than one function—should be        considered when selecting a gene target for genetic        manipulation. Many proteins that moonlight are enzymes. One        example is Glutamate racemase (Mud) which is a critical enzyme        in cell wall biosynthesis but also plays a role in gyrase        inhibition. Owing to its multifunctional character, the        usefulness of Murl-targeted strategies cannot be predicted        unless mutations in these genes are obtained and evaluated for        the impact on the bacterial cell physiology.    -   e. In addition, the type of immune response elicited by a        vaccine may not be appropriate to provide an adequate protection        against infection (vaccine failure). The specific requirements        for an effective vaccine will vary according to the nature of        the pathogen. In the case of extracellular pathogens, the major        antibodies provide adaptative mechanisms for the defense of the        organism, while the presence of T cells is essential in        controlling intracellular organisms. In consequence, live        attenuated vaccines serve as better immunogens that killed        bacteria or subunit compositions by means of simple        multiplication, as well as by the modifications of bacterial        antigens that occur during in vivo infection. Thereby, a live        attenuated strain could engender a broader and adequate immune        response, especially in the intracellular phase. In this sense,        gene-targeted strategies of attenuation should be carefully        tested in vaccine candidates, as the ability of the manipulated        bacteria to exploit the natural pathways of infection could be        potentially impaired and not trigger a broadly protective immune        response.    -   f. In addition, irrespectively of the attenuation level or the        type of immune response elicited, the number of doses        administrated to achieve an acceptable level of protection with        a specific GMO (effective and lasting) can be unsustainable for        a vaccine schedule.    -   g. Furthermore, the route of administration of a vaccine can        determine the type of immune response mounted and to be crucial        for its success. Depending on the route of administration, the        vaccine may enter the organism in different ways: skin (in this        case the antigen is taken up by Langerhans cells that act as        antigen-presenting cells in the T-zone of regional lymph nodes);        mucosa (here the capture of antigen is carried out mainly by M        cells and the immune response is developed in the Peyer's        patches) or blood (the antigen would target the spleen where it        would be processed by splenic macrophages). Consequently, once        attenuation level is established for a GMO, the site of vaccine        administration could determine the failure or success of        vaccination. In this regard, it has ben demonstrated that        intramammary but not intraperitoneally administration of a live        attenuated S. aureus strain significantly decreases the        bacterial load in mamary glands after challenge with the wild        type strain. The proposed vaccine candidate, S. aureus 8325-4        A523, is a temperature-sensitive mutant isolated after        mutagenesis with nitrosoguanidine, which replicates well at low        temperatures (below 32° C.) but undergoes a limited number of        divisions when tranferred to the mammalian body temperature. The        authors performed challenge experiments with the S. aureus        8325-4 wild type strain to compare bacterial loads in mammary        glands between vaccinated and non-vaccinated animals as measure        of vaccine protection efficacy. These authors concluded the        following: “The number of S. aureus CFU recovered from the        mammary glands of mice immunized by the intramammary route was        significantly lower (7×10² CFU) than that found in control mice        (1.5×10⁵ CFU). Conversely, the number of CFU recovered from        mammary glands of mice immunized by any of the intraperitoneal        protocols was as high as that recovered form control mice glands        (P>0.5)”. Therefore, even with a potentially good candidate, the        route of administration can determine the efficacy of the live        attenuated mutant as a vaccine.    -   h. Lastly, a vaccine can be unable to induce cross-reactive        antibodies against multiple strains of the same bacterial        species. Although live attenuated strains can elicit antibodies        that are protective in animal models, this protection is        generally seen only when the parental strain used to create the        vaccine strain is also used in the challenge studies.        Broad-based protection against other strains usually is not        reliable generated or tested. Moreover, antibodies produced,        although adequately elicited and cross-reactive, may not last        long nor be protective in a model of challenge with the wild        type pathogen.

In summary, a live vaccine should be sufficiently attenuated (ora-virulent) to avoid unacceptable pathological effects, but on the otherhand it must elicit an adequate immune response capable of conferring alasting protection in the host against the disease (protective immunity)independently of the bacterial strain.

Demonstrating that a live vaccine is sufficiently attenuated (ora-virulent) to avoid unacceptable pathological effects and elicits anadequate immune response capable of conferring a lasting protection inthe host against the disease (protective immunity) independently of thebacterial strain, is not an easy task. In this sense, WO99/25376describes a method of eliciting a T cell immune response against anantigen in a mammal which comprises administering to said mammal anauxotrophic attenuated strain of Listeria which expresses the antigen.Said auxotrophic attenuated strain is described therein as having amutation in at least one gene whose protein product is essential forgrowth of the Listeria. In particular, the invention describes anauxotrophic attenuated strain for the synthesis of D-alanine whichfurther comprises DNA encoding a heterologous antigen, wherein theheterologous antigen is preferably an HIV-1 antigen.

In WO99/25376, the results are presented as showing that the auxotrophicstrain of Listeria provides protection against challenge by L.monocytogenes in BALB/c mice making this strain alledgely suitable foruse in a vaccine composition for protection against an infection causedby this organism. However, the experimental examples provided thereinmerely establish that attenuated auxotrophic D-alanine mutants of L.monocytogenes elicit a CTL (host cytotoxic T cell) response. Anantibody-mediated immune response (humoral immunity) is not consideredtherein nor are results provided in this sense. Moreover, the protectioneffect of this mutant is therein determined by measuring the bacterialcounts in the spleen of infected mice after challenge with the wild typeListeria. In this sense, the effectiveness of a vaccine against acuteand lethal bacterial infections (especially those causing sepsis) canonly be asessed if survival assays are conducted. In addition, mutantListeria injected without D-alanine are described therein as providinglittle protection. In contrast, when D-alanine was supplemented in theinitial inoculum of the mutant organism to achieve the same protectionas the wild type strain (at the time of initial immunization), this hadthe effect of reducing the lethal dose of the mutant about 10 fold, aserious limitation for the safety of this mutant if the lost ofattenuation is considered. Therefore, the results provided in WO99/25376for the D-alanine mutants therein described, fail to demonstrate theuselfulness of these mutant strains as vaccine candidates againstextracellular bacterial pathogens and acute systemic infections.Moreover, the lack of cross-protection data with these mutants does notassure the effectiveness of a vaccine composed of D-alanine auxotrophsto generate a broadly protective immune response against other L.monocytogenes strains, much less its usefulness for generating vaccinecandidates in other bacterial species.

In addition, in the detailed description of WO99/25376, the inventorsmake the following suggestion: “Additional potential useful targets forthe generation of additional include the genes involved in the synthesisof the cell wall component D-glutamic acid. To generate D-glutamic acidauxotrophic mutants, it is necessary to inactivate the dat gene, whichis involved in the conversion of D-glu+pyr to alpha-ketoglutarate+D-alaand the reverse reaction. It is also necessary to inactivate theglutamate racemase gene, dga”. However, one of ordinary skill in the artwill know that there is no reasonable expectation of success in light ofthe information presented therein that D-glutamic acid auxotrophicstrains of Listeria can presumably confer a satisfactory level ofattenuation to avoid unacceptable pathological effects and elicit anadequate immune response capable of conferring a lasting protection inthe host against the disease (protective immunity). As discussed above,each gene-targeted strategy should be evaluated case by case. Moreover,the glutamate racemase enzyme has moonlinghtening functions that canaffect celular viability if its coding genes are manipulated. In thissense, the use of glutamate racemase as a target to generate D-glutamateauxotrophic vaccine strains to confer protection against bacterialinfections cannot be extrapolated from the previous invention, becausethere is no sustained evidence presented, and it is not obvious thatsuch a strain can be immunogenic.

The latter statement, namely that it is not obvious that suchD-glutamate auxotrophic vaccine strain can be immunogenic, is furthersustained by the fact that there are no studies nor inventionsdemonstrating the ability of D-glutamate auxotrophic micro-organisms tobe useful as live vaccines for conferring protection againstbacterial-caused diseases in the current state of vaccine development.In this sense, and despite the considerable level of attenuation of aD-glutamic acid auxotrophic mutant demonstrated in M. P. Cabral et al,“Blockade of glutamate racemisation during cell-wall formation preventsbiofilm and proliferation of Acinetobacter baumannii in vivo”, Abstractof the 23^(rd) ESCMID congress (European Society of ClinicalMicrobiology and Infectious Diseases) held in Berlin from the 27^(th) tothe 30^(th) of April, 2013 (the only reference showing a relationbetween D-glutamate auxotrophy and in vivo loss of virulence), it isnoted that one of the main obstacles to the development of vaccines isthe difficulty in achieving a satisfactory level of attenuation withoutseverely compromising immunogenicity (protection). So, correlationbetween attenuation and protection need to be invariably tested for eachgene-targeted modification strategy in order to develop an effectivevaccine against bacterial infections. In this sense, the above mentioneddocument (M. P. Cabral et al, “Blockade of glutamate racemisation duringcell-wall formation prevents biofilm and proliferation of Acinetobacterbaumannii in vivo”, Abstract of the 23^(rd) ESCMID congress (EuropeanSociety of Clinical Microbiology and Infectious Diseases) held in Berlinfrom the 27^(th) to the 30^(th) of April, 2013), even thought itdescribes the attenuation of an Acinetobacter baumannii straincharacterized by the in-frame deletions of glutamate racemase genes,fails to provide any data showing the protective efficacy of such astrain against A. baumannii infections. Providing data showing theprotective efficacy of an attenuated strain is crucial to determine theusefulness of such a strain as a vaccine as demonstrated in thefollowing prior art documents.

In M. K. Hondalus et al, “Attenuation of and protection induced by aleucine auxotroph of Mycobacterium tuberculosis”, Infection and Immunity68 (2000) 2888-2898, a leucine auxotroph of M. tuberculosis was createdby allelic exchange so that the mutant was unable to replicate inmacrophages (proving that the bacteria was attenuated). In fact, FIG. 5of this document shows how mice infected with the leucine auxotroph ofM. tuberculosis had a 100% survival rate 22 weeks post-infection(establishing that leucine auxotroph was indeed attenuated). However,the leucine auxotrophic mutant was shown to be less effective than thelive BCG vaccine in reducing organ burdens and tissue pathology ofBALB/c mice challenged intravenously. This document illustrates that itis not enough to have an attenuated strain to have a vaccine and thatimmunogenicity is a key issue.

Furthermore, in M. S. Jr Pavelka et al, “Vaccine efficacy of a lysisneauxotroph of Mycobacterium tuberculosis”, Infection and Immunity 71(2003) 4190-4192, it was demonstrated that a single intravenousimmunization of mice with the M. tuberculosis mutant (a lysine auxotrophof M. tuberculosis) did not generate a significant protective responseto the subsequent aerosol and that a single immunization with theauxotroph was insufficient for reducing the bacterial burden in thelungs and spleens relative to a single immunization with BCG. Only thetriple immunized mice survived as long as the control mice immunizedwith a single dose of BCG. Consequently, again it can be concluded thatimmunogenicity is a key and separate issue from attenuation.

Moreover, prior art reference Ann-Mari Svennerholm et al, “Vaccinesagainst enterotoxigenic Escherichia coli”, Expert review of vaccines 7(2008) 795-804, describes genetically attenuated ETEC (enterotoxigenicEscherichia coli) strains as live oral vectors and characterized assafe. However when evaluating these same strains for protection, neitherthe attack rate for diarrhea nor the total stool volume wassignificantly diminished in vaccines versus placebo recipients.

Lastly, H. K. Kim et al, “Identifying protective antigens ofStaphylococcus aureus, a pathogen that suppresses host immuneresponses”, FASEB J. 25 (2011) 3605-3612, describes whether threeattenuated mutants derived from the Newman strain by transposoninsertional mutagenesis can elicit protective immunity in mice. Thesemutants were constructed in order to block the expression ofexoproteins, surface proteins as well as the processing of surfaceproteins, namely saeR (S. aureus exoprotein), mgrA (multiple generegulator A) and srtA (sortase A). However, mutants lacking saeR ormgrA, despite being attenuated in mice, did not to confer protectiveimmunity to subsequent S. aureus infection.

Consequently, even if a reduced virulence (good level of attenuation)for a particular derivative strain is achieved, its protective capacityin the host must be experimentally assessed to be able to conclude itsusefulness as a live vaccine.

BRIEF DESCRIPTION OF THE INVENTION

A first aspect of the invention refers to a method for the production ofa pharmaceutical composition, preferably a vaccine, comprising mutantlive auxotrophic bacterial strains for D-glutamate, wherein thepharmaceutical composition is suitable for the prophylactic treatment(before infection) and/or therapeutic treatment (after infection orafter the clinical manifestation of the disease caused by the infection)of animals and/or humans against infection with the wild type form ofthe mutant auxotrophic bacteria of the composition, and wherein saidpharmaceutical composition is produced by a method comprising the stepsof:

-   -   a. obtaining mutant live auxotrophic bacterial strains for        D-glutamate;    -   b. introducing said mutant live auxotrophic bacerial strains in        a pharmaceutically acceptable carrier or diluent and optionally        adding an adjuvant; and    -   c. Optionally freeze-drying the pharmaceutical composition.

In preferred embodiment of the first aspect of the invention, theproduction method comprises the steps of:

-   -   a. providing a bacterial strain capable of expressing glutamate        racemase and possibly D-amino acid transaminase and comprising a        peptidoglycan cell wall;    -   b. inactivating the gene or genes encoding for the glutamate        racemase enzyme and, if needed, the gene or genes encoding for        the enzyme D-amino acid transaminase in such way that the        bacterial strain is no longer capable of expressing a functional        glutamate racemase and/or a functional D-amino acid        transaminase, wherein the inactivation of said genes thus causes        said bacterial strain to be auxotrophic for D-glutamate; and    -   c. introducing said mutant live auxotrophic bacterial strains in        a pharmaceutically acceptable carrier or diluent and optionally        adding an adjuvant; and    -   d. Optionally freeze-drying the pharmaceutical composition.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the pharmaceutical composition is avaccine and the production method comprises adding an adjuvant.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the bacterial strain of step a) isa gram positive or gram negative bacteria. Preferably, the bacterialstrain of step a) is selected from the list of bacterial speciesconsisting of: Acinetobacter baumannii, Acinetobacter baylyi,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacterjunii, Acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacterpittii, Acinetobacter radioresistens, Actinobacillus lignieresii,Actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonasveronii subsp. sobria, Aggregatibacter actinomycetemcomitans, Arcobacterbutzleri, Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillusanthracis, Bacillus bataviensis, Bacillus cellulosilyticus, Bacilluscereus, Bacillus clausii, Bacillus licheniformis, Bacillus megaterium,Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallel, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.

More preferably, said bacterial strain of step a) is selected from thelist consisting of the following species: Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus. Still more preferably,the bacterial strain is the bacterial strain of A. baumannii designatedAcinetobacter baumannii Delta0380/Delta3398 and deposited under theBudapest treaty before the Spanish Type Culture Collection on Apr. 14,2014 with strain number 8588 by Fundación Profesor Novoa Santos. Stillmore preferably, the bacterial strain is the bacterial strain of P.aeruginosa designated Pseudomonas aeruginosa DeltaPA4662 and depositedunder the Budapest treaty before the Spanish Type Culture Collection onApr. 14, 2014 with strain number 8589 by Fundación Profesor NovoaSantos. Still more preferably, the bacterial strain is the bacterialstrain of S. aureus designated 132deltamurl/deltadat and deposited underthe Budapest treaty before the Spanish Type Culture Collection on Jun.11, 2014 with strain number 8587 by Fundación Profesor Novoa Santos.

A second aspect of the invention refers to a pharmaceutical composition,preferably a vaccine, comprising mutant live auxotrophic bacerialstrains for D-glutamate and a pharmaceutically acceptable carrier ordiluent and optionally an adjuvant, wherein said pharmaceuticalcomposition is suitable for the prophylactic (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

In a preferred embodiment of the second aspect of the invention, saidpharmaceutical composition is a vaccine and said vaccine optionallycomprises an adjuvant.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutically acceptablecarrier or diluent is selected from the list consisting of water,culture fluid, a solution of physiological salt concentration and/orstabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol,starch, sucrose, glucose, dextran), proteins such as albumin or casein,protein containing agents such as bovine serum or skimmed milk andbuffers (e.g. phosphate buffer).

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said adjuvant is selected from thelist consisting of Freunds Complete and Incomplete adjuvant, vitamin E,non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulatingcomplexes), Saponins, mineral oil, vegetable oil, Carbopol, the E. coliheat-labile toxin (LT) or Cholera toxin (CT), aluminium hydroxide,aluminium phosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F®or Marcol 52®, saponins and vitamin-E solubilisate.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutical compositioncomprises a dose of mutant live auxotrophic bacterial strains forD-glutamate ranging between 10³ and 10¹⁰ bacteria.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutical composition isin a freeze-dried form.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, the bacterial strain is selectedfrom the list of bacterial species consisting of: Acinetobacterbaumannii, Acinetobacter baylyi, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter lwoffii,Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacterradioresistens, Actinobacillus lignieresii, Actinobacillus suis,Aeromonas ca viae, Aeromonas hydrophila, Aeromonas veronii subsp.sobria, Aggregatibacter actinomycetemcomitans, Arcobacter butzleri,Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillus anthracis,Bacillus bataviensis, Bacillus cellulosilyticus, Bacillus cereus,Bacillus clausii, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella muftocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.

More preferably, said bacterial strain of step a) is selected from thelist consisting of the following species: Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus. Still more preferably,the bacterial strain is the bacterial strain of A. baumannii designatedAcinetobacter baumannii Delta0380/Delta3398 and deposited under theBudapest treaty before the Spanish Type Culture Collection on Apr. 14,2014 with strain number 8588 by Fundación Profesor Novoa Santos. Stillmore preferably, the bacterial strain is the bacterial strain of P.aeruginosa designated Pseudomonas aeruginosa DeltaPA4662 and depositedunder the Budapest treaty before the Spanish Type Culture Collection onApr. 14, 2014 with strain number 8589 by Fundación Profesor NovoaSantos. Still more preferably, the bacterial strain is the bacterialstrain of S. aureus designated 132deltamurl/deltadat and deposited underthe Budapest treaty before the Spanish Type Culture Collection on Jun.11. 2014 with strain number 8587 by Fundación Profesor Novoa Santos.

A third aspect of the invention refers to a mutant live auxotrophicbacterial strain for D-glutamate, wherein said bacterial strain is thebacterial strain of P. aeruginosa designated Pseudomonas aeruginosaDeltaPA4662 and deposited under the Budapest treaty before the SpanishType Culture Collection on Apr. 14, 2014 with strain number 8589 byFundacion Profesor Novoa Santos.

A third aspect of the invention also refers to a mutant live auxotrophicbacterial strain for D-glutamate, wherein said bacterial strain is thebacterial strain of S. aureus designated 132deltamurl/deltadat anddeposited under the Budapest treaty before the Spanish Type CultureCollection on Jun. 11, 2014 with strain number 8587 by FundaciónProfesor Novoa Santos.

A fourth aspect of the invention refers to the bacterial strain asdefined in the third aspect of the invention, for use as a medicament,in particular for use as a vaccine.

A fifth aspect of the invention refers to the pharmaceutical compositionof the second aspect of the invention or the mutant live auxotrophicbacterial strain for D-glutamate of the third or fourth aspects of theinvention, for use in a method of prophylactic treatment (beforeinfection) and/or therapeutic treatment (after infection or after theclinical manifestation of the disease caused by the infection) ofanimals and/or humans against infection with the wild type form of themutant auxotrophic bacteria of the composition.

A sixth aspect of the invention refers to an antibody or fragmentthereof selected from the group consisting of Fab, F(ab′)2, Fv, scFv,di-scFv and sdAB, capable of recognizing a mutant live auxotrophicbacterial strain for D-glutamate, wherein said antibody or fragmentthereof is suitable for the prophylactic treatment (before infection)and/or therapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

A seventh aspect of the invention refers to an antibody or fragmentthereof selected from the group consisting of Fab, F(ab′)2, Fv, scFv,di-scFv and sdAB, obtained or obtainable after immunization of a mammalwith a mutant live auxotrophic bacterial strain for D-glutamate, whereinsaid antibody or fragment thereof is suitable for the prophylactictreatment (before infection) and/or therapeutic treatment (afterinfection or after the clinical manifestation of the disease caused bythe infection) of animals and/or humans against infection with the wildtype form of the mutant auxotrophic bacteria of the composition

An eighth aspect of the invention refers to a pharmaceuticalcomposition, preferably a vaccine, comprising the antibodies orfragments thereof of any of the sixth or seventh aspects of theinvention and a pharmaceutically acceptable carrier or diluent andoptionally an adjuvant, wherein said pharmaceutical composition issuitable for the prophylactic (before infection) and/or therapeutictreatment (after infection or after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacteria ofthe composition. In a preferred embodiment of the eighth aspect of theinvention, said pharmaceutical composition is a vaccine wherein saidvaccine optionally comprises an adjuvant.

A ninth aspect of the invention refers to the antibodies or fragmentsthereof of the sixth or seventh aspects of the invention, for use intherapy, in particular for use in passive immunization.

A tenth aspect of the invention refers to the pharmaceutical compositionof the eighth aspect of the invention or the antibodies or fragmentsthereof of any of the sixth or seventh aspects of the invention, for usein a method of prophylactic treatment (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

An eleventh aspect of the invention refers to the pharmaceuticalcomposition of the second or eighth aspects of the invention or themutant live auxotrophic bacterial strain for D-glutamate of the thirdaspect of the invention or the antibodies or fragments thereof of any ofthe sixth or seventh aspects of the invention, for use in a method ofprophylactic treatment (before infection) and/or therapeutic treatment(after infection or after after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacteria ofthe composition and wherein said composition, bacterial strain orantibody or fragment thereof is administered intranasally,intradermally, subcutaneously, orally, by aerosol, intramuscularly, wingweb and eye-drop administration.

In addition, the authors of the present invention have surprisinglyfound that by using a kit or device comprising an antibody or fragmentthereof of the invention, the kit permits a reliable qualitative and/orquantitative analysis of bacterial species in a biological of sample ofa subject and, in particular, in the plasma of subjects suspected ofsuffering from a disease of bacterial origin.

Therefore, a twelfth aspect of the invention refers to a kit or devicecomprising the antibody or fragment thereof of any of the seventh oreigth aspect of the invention.

A preferred embodiment of the twelfth aspect of the invention refers toa kit or device for detecting an infection of bacterial origin throughan immunoassay comprising:

-   -   (i) a first antibody called “capture antibody” as defined in any        of the sixth or seventh aspects of the invention, wherein said        first antibody is preferably attached to a solid support;    -   (ii) a second labeled antibody called “detection antibody” which        recognizes a region other than the region recognized by the        first antibody , wherein said second antibody comprises a marker        which may be fluorescent , luminescent or an enzyme;    -   (iii) a reagent showing affinity for the second antibody, said        reagent being coupled to a first member of a binding pair; and    -   (iv) a second member of a binding pair coupled to a fluorescent,        luminescent or an enzyme, wherein the binding pair is selected        from the group consisting of: hapten and antibody; antigen and        antibody; biotin and avidin; biotin and streptavidin; a biotin        analogue and avidin; a biotin analogue and streptavidin; sugar        and lectin; an enzyme and a cofactor; a nucleic acid or a        nucleic acid analogue and the complementary nucleic acid or        nucleic acid analogue.

A thirteenth aspect of the invention refers to the use of the kit ordevice of the twelfth aspect of the invention, for the qualitativeand/or quantitative determination of bacterial species or bacterialstrains in a biological sample from a mammal, in particular, in theplasma of a mammal suspected of suffering from a bacterial disease.

A fourteenth aspect of the invention refers to a method of cultivationof bacterial strains auxotrophic for D-glutamate comprising theutilization of a concentration of D-glutamate between 0.00001 and 120mM. Preferably, the concentration range of D-glutamate is between0.01-50 mM. More preferably, the concentration range of D-glutamate isbetween 10-20 mM.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically shows the general structure of the bacterial wallof a gram positive bacterium (non-depicted teichoic acids and proteins).PG: peptidoglycan (murein). M: cytoplasmic membrane.

FIG. 2 schematically shows the structure and constitution of thebacterial wall of a gram negative bacterium (non-depictedlipopolysaccharides and proteins). PG: peptidoglycan (murein). OM: outermembrane. IM: inner membrane.

FIG. 3 schematically shows the sequence of metabolic processesculminating in the formation of D-glutamate and the incorporationthereof in the bacterial cell wall peptidoglycan. The dat, murl and murDgenes depicted in the figure encode the Dat, Murl and MurD proteins,respectively.

FIG. 4 shows the alignment of the amino acid sequences of the two Murlglutamate racemases of A. baumannii ATCC 17978 (A3MIP5_ACIBT andA3MA43_ACIBT), Escherichia coli (MURI_ECOLI) and P. aeruginosa(MURI_PSEAE) using the Clustal Omega program. Identical residues in allthe glutamate racemases are depicted with a dark gray background.

FIG. 5 shows PCR confirmation of the deletions in Acinetobacterbaumannii ATCC 17978 mutants Δ0380, Δ3398 and Δ0380/Δ3398. A:Oligonucleotides EXTFW0380 and EXTRV0380 were used to generate fragmentswith 1116 bps from strains carrying wild type locus A1S_0380 or a 345 byfragment from strains carrying mutant locus Δ0380. B: OligonucleotidesEXTFW3398 and EXTRV3398 were used to generate fragments with 1056 bpsfrom strains carrying wild type locus A1S_3398 or a 516 bp fragment fromstrains carrying mutant locus Δ3398. The DNA fragments in each lane havethe following matchups: MW, molecular weight pattern; 0380, amplicongenerated from the strain carrying wild type locus A2S_0380; 3398,amplicon generated from the strain carrying wild type locus A1S_3398;Δ0380/Δ3398, amplicon generated from the defective strain in the twoloci A1S_0380 and A1S_3398; Δ3398 and Δ0380, amplicons generated fromthe defective strains in loci A1S_3398 and A1S_0380, respectively.

FIG. 6 shows the screening of the colonies resulting from theco-integrants during construction of the A. baumannii ATCC 17978 doublemutant Δ0380/Δ3398. The individual colonies were selected from LB agarwith 15% sucrose and 10 mM D-glutamate and inoculated in the sameposition in LB agar plates with and without 10 mM D-glutamate. Colonieswith the Δ0380/Δ3398 genotype grow exclusively in plates withD-glutamate; colonies with the Δ0380 genotype grow with and withoutD-glutamate.

FIG. 7 shows the growth and viability assays of A. baumannii wild typestrain ATCC 17978 and of double mutant strain Δ0380/Δ3398. StrainΔ0380/Δ3398 shows normal growth in culture medium supplemented with 10mM of D-glutamate but is unable to grow without the exogenous supply ofthis compound. In contrast, the wild type strain grows as per normal inLB medium with and without the addition of D-glutamate. In each panel,the solid squares (▪) represent wild type strain in medium withD-glutamate; empty squares (□) represent the wild type strain withoutD-glutamate; solid circles () represent the double mutant in mediumwith D-glutamate and empty circles (◯) represent the double mutant inmedium without D-glutamate. A: Double mutant and wild type strainculture optical density. B: Double mutant and wild type strain cultureviability (Log₁₀ CFU/mL).

FIG. 8 shows the differences at the cell division and morphology levelin the A. baumannii ATCC 17978 Δ0380/Δ3398 double mutant with respect tothe wild type homologue thereof in the presence of differentconcentrations of D-glutamate.

Microphotographs were taken with a scanning electron microscope. A: Bothstrains were cultured at increasing concentrations of D-glutamate andthe microphotographs were taken on the same scale (the bar indicates ascale of 10 μm); B: Both strains were cultured in the presence of 0.1 mMD-glutamate, and the microphotographs were taken progressively on scalesof a decreasing order.

FIG. 9 shows different atypical morphologies, progressive degenerationof the cell wall and lysis of the A. baumannii ATCC 17978 mutant strainΔ0380/Δ3398 when kept in the absence of D-glutamate. Microphotographswere taken with a transmission electron microscope on different scales.Black arrows indicate intact bacterial cells with normal morphology oratypical cell division; dashed arrows indicate fragmented cells withoutbacterial cell wall, lysed cells or disorganized internal content,dispersed genetic material, aggregation of membranes and/or liposomes.

FIG. 10 shows the percentage of survival of BALB/c mice afterintraperitoneal injection with different doses of A. baumannii wild typestrain ATCC 17978 (A) and Δ0380/Δ3398 mutant strain (B) (n=6) todetermine the lethal dose (LD) for which 100% of susceptible mice willdie (LD₁₀₀) (A) LD₁₀₀=2.5×; (B) LD₁₀₀=6×. Mice survival was monitoredduring 7 days.

FIG. 11 shows the bacterial load in the liver of BALB/c mice (n=8-9mice) 12 hours post-infection with a 2× dose of the A. baumannii wildtype strain ATCC 17978, strain Δ0380, strain Δ3398 and strainΔ0380/Δ3398. P-value according to the Mann-Whitney U test. Each dotrepresents the individual bacterial load of the liver of a mouse andeach horizontal line represents the respective average for each group.

FIG. 12 shows the bacterial load in the liver, spleen and lungs ofBALB/c mice (n=10) 12 hours post-infection with a 4× dose of the A.baumannii wild type strain ATCC 17978 administered on day 21, after themice were pre-immunized on days 0 and 14 with strain Δ0380/Δ3398, ornon-immunized (saline control). P-value according to the Mann-Whitney Utest. Each dot represents the individual bacterial load of the organ ofa mouse. The average value of each group is represented by a horizontalline.

FIG. 13 shows the Log₁₀ 1/Endpoint titer of IgG antibodies producedagainst the A. baumannii strain ATCC 17978 in BALB/c mice (n=12) onpost-vaccination days 7 and 21, and in non-vaccinated control mice(saline control). The antibody titers were determined by indirect ELISA.*P<0.0001 compared with the group of non-vaccinated mice; #P<0.0240compared with the production of IgGs on post-vaccination day 7; P-valueaccording to the Mann-Whitney U test. The boxes represent the first andthird quartiles; the horizontal line represents the median; the whiskersrepresent the range.

FIG. 14 shows the Log₁₀ 1/Endpoint titer of IgG antibodies producedagainst A. baumannii strain ATCC 17978 in BALB/c mice (n=6) onpost-vaccination day 21 with different doses of A. baumannii strain ATCC17978 (0.01×; 0.05×; 0.1×; 0.5× and 1×) and in the non-vaccinatedcontrol mice (0×). The antibody titers were determined by indirectELISA. *P<0.05 compared with the group of non-vaccinated mice; P-valueaccording to the Mann-Whitney U test. The boxes represent the first andthird quartiles; the horizontal line represents the median; the whiskersrepresent the range.

FIG. 15 shows the cross-reactivity (titer) of IgG antibodies produced byBALB/c mice on post-vaccination day 21 and in non-vaccinated controlmice (saline control) against three different A. baumannii strains: ATCC17978, ATCC 19606 and AbH12O-A2.

FIG. 16 is the percent survival of BALB/c mice (n=12) followingintraperitoneal infection with a 4× dose of A. baumannii ATCC 17978 wildtype strain. Vaccinated mice were immunized on days 0 and 14 with A.baumannii Δ0380/Δ3398 strain and infected with the wild type strain atday 21. Non-vaccinated mice were administered saline on days 0 and 14and infected with the wild type strain at the same day. *P<0.0001survival of vaccinated group compared to control group. P-value,according to the Mantel-Cox test (log-rank test).

FIG. 17 is the percent survival of BALB/c mice (n=9) followingintraperitoneal infection with a 4× dose of A. baumannii AbH12O-A2strain. Vaccinated mice were immunized on days 0 and 14 with A.baumannii Δ0380/Δ3398 strain and infected with the clinical strain atday 21. Non-vaccinated mice were administered saline on days 0 and 14and infected with the clinical strain at the same day. *P<0.0001survival of vaccinated group compared to control group. P-value,according to the Mantel-Cox test (log-rank test).

FIG. 18 is the percent survival of BALB/c mice (n=6-8) followingintraperitoneal infection with a 0.75× dose of the capsulated A.baumannii Ab307-0294 strain. Vaccinated mice were immunized on days 0and 14 with A. baumannii Δ0380/Δ3398 strain and infected with the highlyvirulent strain at day 21. Non-vaccinated mice were administered salineon days 0 and 14 and infected with Ab307-0294 strain at the same day.*P=0.0022 survival of vaccinated group compared to control group.P-value, according to the Mantel-Cox test (log-rank test).

FIG. 19 is the Log₁₀ CFU/mL of recovered A. baumannii ATCC 17978 wildtype and Δ0380/Δ3398 strains when grown in distilled water at 37° C.with agitation (180 rpm) during 40 days. CFU's were determined bycounting colonies plated onto LB (wild type strain) and LB supplementedwith 10 mM D-glutamate (mutant strain). All cultures were performed intriplicate.

FIG. 20 is the number of A. baumannii Δ0380/Δ3398 colonies (CFU/mL)recovered from LB (◯) and LB supplemented with 10 mM D-glutamate ()when this strain was cultivated onto LB supplemented with 10 mMD-glutamate at 37° C. with agitation (180 rpm) during 8 days.

FIG. 21 shows the number of A. baumannii colonies (Log₁₀) CFU/mL)recovered from the blood of mice inoculated with 100 μL A. baumanniiATCC 17978 wild type and Δ0380/Δ3398 strains (1× dose) along the time.

FIG. 22 shows the screening of resolved co-integrants duringconstruction of the ΔPA4662 mutant of P. aeruginosa.

FIG. 23 illustrates the PCR confirmation of deletions in ΔPA4662 mutantof P. aeruginosa. Primers EXTFWPA4662 and EXTRVPA4662 were used togenerate a 1741 by fragment from strains carrying the wild type PA4662allele or a 943 bp fragment from strains carrying the ΔPA4662 allele.Lane labels and samples analyzed are as follows: MW: DNA ladder;ΔPA4662: amplicon from strain carrying the ΔPA4662 mutant allele; andPA4662: amplicon from strain carrying the wild type PA4662 allele.

FIG. 24 shows the percentage of survival of BALB/c mice afterintraperitoneal injection with different doses of P. aeruginosa wildtype strain PAO1 (A) and ΔPA4662 mutant strain (B) (n=4) to determinethe lethal dose (LD) for which 100% of susceptible mice will die (LD₁₀₀)(A) LD₁₀₀=0.4×; (B) LD₁₀₀>40×. Mice survival was monitored during 7days.

FIG. 25 shows the atypical morphology and the progressive degenerationof the cell wall and lysis of the P. aeruginosa mutant strain ΔPA4662when maintained in the absence of D-glutamate. A-E: MH media; F-I: LBmedia; J-T: LB+MgCl₂ (30 mg/L)+CaCl₂ (75 mg/L). Micrographs were takenwith a transmission electron microscope at different scales. Blackarrows indicate intact bacterial cells with normal morphology oratypical cell division; dashed arrows indicate fragmented cells withoutbacterial cell wall, lysed cells or disorganized internal content,dispersed genetic material, aggregation of membranes and/or liposomes.

FIG. 26 is the percent survival of BALB/c mice (n=8) followingintraperitoneal infection with a 0.4× dose of P. aeruginosa PAO1 wildtype strain. Vaccinated mice were immunized on days 0 and 14 with P.aeruginosa ΔPA4662 strain (0.4× dose—A; 0.04× dose—B) and infected withthe wild type strain at day 25. Non-vaccinated mice were administeredsaline on days 0 and 14 and infected with the wild type strain at thesame day. *P<0.0001 survival of vaccinated group compared to controlgroup. P-value, according to the Mantel-Cox test (log-rank test).

FIG. 27 is the percent survival of BALBIc mice (n=8) followingintraperitoneal injection of a 0.4× dose of P. aeruginosa PAO1 wild typestrain. In (A), mice were passively immunized with vaccine serum(generated with the ΔPA4662 vaccine) or administrated naïve serum priorto infection. In (By, mice were administered two dosis of vaccine serum(generated with the ΔPA4662 strain) or naïve serum after the developmentof an acute sepsis symptoms. *P<0.05 survival of mice passivelyimmunized with vaccine serum compared to mice receiving naïve serum.P-value, according to the Mantel-Cox test (log-rank test).

FIG. 28 is the Log₁₀ CFU/mL of recovered P. aeruginosa PAO1 wild typeand ΔPA4662 strains when grown in distilled water at 37° C. withagitation (180 rpm) during 157 days. CFU's were determined by countingcolonies plated onto LB (wild type strain) and LB with 10 mM D-glutamate(mutant strain). All cultures were performed in triplicate.

FIG. 29 is the number of P. aeruginosa ΔPA4662 colonies (CFU/mL)recovered from LB (◯) and LB supplemented with 10 mM D-glutamate ()when this strain was cultivated onto LB with 10 mM D-glutamate at 37° C.with agitation (180 rpm) during 5 days.

FIG. 30 shows the screening of the colonies resulting from the singlemutant Amurl after the second crossover event during construction of theS. aureus double mutant Δmurl/Δdat. The individualerythromycin-sensitive colonies were selected from TSB agar platessupplemented with X-Gal (150 μg/mL) and inoculated in the same positionin TSB agar plates with and without 10 mM D-glutamate. Colonies with theΔmurl/Δdat genotype grow exclusively in plates with D-glutamate;colonies with the Δmurl genotype (lacking the murl gene but with anintact copy of the dat gene) grew properly with and without D-glutamate.

FIG. 31 is the PCR confirmation of the deletions in S. aureus 132mutants Δmurl, Δdat and Δmurl/Δdat. The primers used were A:murlF/murlR; B: murlExtF/murlExtR; C: murlseqF/murlseqR; D: datF/datR;E: datExtF/datExtR; and F: datseqF/datseqR. Lanes: MM, molecular markerGeneRuler 1 kb; Δmurl, fragments obtained from strains carrying mutantlocus Δmurl; Δdat, fragments obtained from strains carrying mutant locusΔdat; Δmurl/Δdat, fragments obtained from strains defective in the twoloci Δmurl and Δdat; wild type, fragments obtained from S. aureus 132wild type strain carrying two loci, murl and dat.

FIG. 32 shows the growth and viability assays of S. aureus 132 wild typeand of double mutant strain Δmurl/Δdat. Strain Δmurl/Δdat shows normalgrowth in TSB supplemented with 20 mM of D-glutamate but is incapable togrow without the exogenous supply of this compound. In contrast, thewild type strain grows in TSB with and without the addition ofD-glutamate. Each symbol represents one strain as indicated in thelegend. A: Culture turbidity; B: Culture viability.

FIG. 33 shows alterations at morphological level in the S. aureus 132double mutant Δmurl/Δdat with respect to the S. aureus wild type strainthereof in the presence of different concentrations of D-glutamate.Images were taken with a scanning electron microscope. A: both strainswere incubated at increasing concentrations of D-glutamate and themicrophotographs were taken on the same scale (horizontal bars indicatea scale of 10 μm); B: both strains were cultured in medium supplementedwith 0.1 mM D-glutamate and the images were taken progressively onscales of a decreasing order.

FIG. 34 shows different atypical morphologies, progressive degenerationof the cell wall and lysis of the S. aureus 132 double mutant strainΔmurl/Δdat when kept in the absence of D-glutamate. Images were takenwith a transmission electron microscope on different scales as specifiedby horizontal bars. Black arrows indicate intact bacterial cells withnormal morphology or atypical cell division; dashed arrows indicatefragmented cells without bacterial cell wall, lysed cells ordisorganized internal content, dispersed genetic material, aggregationof membranes and/or liposomes.

FIG. 35 shows the percentage of survival of BALB/c mice afterintraperitoneal injection with different doses of S. aureus 132 wildtype (A) and Δmurl/Δdat mutant (B) (n=3-4) to determine the lethal dose(LD) for which 100% of susceptible mice will die (LD₁₀₀) (A) LD₁₀₀=3×;(B) LD₁₀₀>30×. Mice survival was monitored during 14 days.

FIG. 36 shows the bacterial load in the spleen (A) and blood cultures(B) of BALB/c mice (n=4-6) 20 hours post-infection with a 5× dose of theS. aureus 132 wild type strain after mice were pre-immunized on days 0and 14 with a 10× dose of strain Δmurl/Δdat, or non-immunized (salinecontrol). Each dot represents the individual bacterial load of spleen ofa mouse. The average value of each group is represented by a horizontalline. P=0.0095 compared with the group of non-vaccinated mice. P-valueaccording to the Mann-Whitney U test. Blood cultures from each mousewere incubated 18 hours at 37° C. without shaking: (+), positive; (−),negative.

FIG. 37 shows bacterial load in the spleen (A) and blood (B) of BALB/cmice (n=8-9) 22 hours post-infection with a 5× dose of S. aureus 132wild type strain. Mice were immunized on days 0 and 14 with a 8× dose ofstrain Δmurl/Δdat, or non-inmunized (saline control). Each dotrepresents the individual bacterial load of spleen or blood of a mouse.The average value of each group is represented by a horizontal line.P=0.0006 and P=0.0002 compared with the group of non-vaccinated mice.P-value according to the Mann-Whitney U test.

FIG. 38 is the percent survival of BALB/c mice (n=10-13) followingintraperitoneal infection with a 5× dose of S. aureus 132 wild typestrain. Vaccinated mice were immunized on days 0 and 14 with a 10× doseof Δmurl/Δdat strain while non-immunized mice were administered salineat the same days. *P=0.031 compared with the group of non-vaccinatedmice. P-value according to Mann-Whitney U test. Mice survival wasmonitored until 96 hours.

FIG. 39 shows the Log₁₀ 1/Endpoint titer of IgG antibodies producedagainst the isogenic S. aureus 132 Δspa strain in BALB/c mice (n=8-10)pre-immunized with a 10× dose of Δmurl/Δdat strain, and in non-immunizedmice (saline control). The antibody titers were determined by indirectELISA. *P<0.0001 compared with the group of non-immunized mice. P-valueaccording to the Mann-Whitney U test. The boxes represent the first andthird quartiles; the horizontal line represents the median; the whiskersrepresent the range.

FIG. 40 shows the cross-reactivity (Log₁₀ 1/Endpoint titer) of IgGantibodies produced by BALB/c mice (n=6-9) pre-immunized with a 10× doseof Amurl/Adat strain, and in non-immunized mice (saline control) againstS. aureus strains of different origin: (A) USA300LAC (human); (B) RF122(bovine); (C) ED133 (ovine); (D) ED98 (poultry). The antibody titerswere determined by indirect ELISA. #P<0.0001 and *P<0.0006 compared withthe group of non-immunized mice. P-value according to the Mann-Whitney Utest. The boxes represent the first and third quartiles; the horizontalline represents the median; the whiskers represent the range.

FIG. 41 shows the Log₁₀ CFU/mL of recovered S. aureus wild type andΔmurl/Δdat strains when grown in distilled water at room temperaturewith agitation (180 rpm) during 5 days. CFU were determined by countingcolonies plated onto TSB agar (wild type) and TSB supplemented with 10mM D-glutamate (double mutant strain). All cultures were performed intriplicate.

FIG. 42 shows the number of S. aureus Δmurl/Δdat colonies (CFU/mL)recovered from TSB (◯) and TSB supplemented with 10 mM D-glutamate ()when this strain was cultivated onto TSB supplemented with 20 mMD-glutamate at 37° C. with agitation (180 rpm) during 11 days.

FIG. 43 shows the number of S. aureus colonies recovered in kidney(CFU/g) (A), spleen (CFU/g) (B), and blood samples (CFU/ml) (C) of mice(n=3/per group) intraperitoneally inoculated with a sub-lethal 0.7× doseof S. aureus 132 wild type strain and a 10× dose of the Δmurl/Δdatstrain, along the time. One mouse (per group/per strain) was euthanizedon post-infection days 1, 2 and 6. CFU/mouse inoculated at time zero isindicated in the Y axis for each strain. Colonies were recovered in TSBagar (wild type strain) or TSB agar plus 10 mM D-glutamate (Δmurl/Δdatstrain).

FIG. 44 shows the endpoint titer Log₁₀ of IgG antibodies producedagainst P. aeruginosa strain PAO1 in BALB/c mice on post-vaccination day40 with different dosis of P. aeruginosa strain PAO1 (0.1×; 0.4×; 1×;4×, 10× and 40×) and in the non-vaccinated control mice (0×). Theantibody titers were determined by indirect ELISA. *P<0.001 comparedwith the group of non-immunized mice; P-value according to unpaired ttest. The boxes represent the first and third quartiles; the horizontalline represents the median; the whiskers represent the range.

FIG. 45 shows the endpoint titer Log₁₀ of IgG antibodies producedagainst the P. aeruginosa strain PAO1 in BALB/c mice (n=5) onpost-vaccination days 7 and 25, and in non-vaccinated control mice. Theantibody titers were determined by indirect ELISA. #P<0.0001 comparedwith the group of non-immunized mice; *P<0.05 comparison betweenpost-vaccination days 7 and 25; P-value according to unpaired t test.The boxes represent the first and third quartiles; the horizontal linerepresents the median; the whiskers represent the range.

FIG. 46 shows the cross-reactivity (titer) of IgG antibodies produced byBALB/c mice on post-vaccination day 34 and in non-vaccinated controlmice (saline control) against multiple P. aeruginosa strains: PAO1,PA28562, PA51430664, PA26132, PAST175, PA29475 and PAl2142.

FIG. 47 shows the bacterial load in the liver, spleen and lungs ofBALB/c mice (n=8) 10 hours post-infection with a 0.4× dose of the P.aeruginosa wild type strain PAO1 administered on day 22, after the micewere pre-immunized on days 0 and 15 with strain ΔPA4662, ornon-immunized (saline control). P-value according to the unpaired ttest. Each dot represents the individual bacterial load of the organ ofa mouse. The average value of each group is represented by a horizontalline.

FIG. 48 shows the Log₁₀ 1/endpoint titer of IgG antibodies producedagainst the A. baumannii strain ATCC 17978 (A), P. aeruginosa strainPAO1 (B) and isogenic S. aureus strain 132 Δspa (C) in BALB/c mice(n=2-3/per dosis/per route) on post-vaccination days 7 (after the 1^(st)vaccine dosis), 21 (after 2^(nd) dosis), 35 (after 3^(rd) dosis), 49(after 4^(th) dosis) and 63 (after 5^(th) dosis, with exceptions) withΔ0380/Δ3398, ΔPA4662 and Δmurl/Δdat vaccine strains, respectively, andin non-vaccinated control mice (saline control). Mice were sequentiallyvaccinated on days 0, 14, 28, 42 and 56 (with exceptions) usingdifferent routes of administration—intraperitoneal, subcutaneous,intramuscular, intranasal—and with different vaccine doses (0.1× and 1×,Δ0380/Δ3398−0.4× and 0.04×, ΔPA4662×0.2×, 1×, 3× and 10×, Δmurl/Δdat).The antibody titers were determined by indirect ELISA. IgG titersproduced in vaccinated mice were compared with the group ofnon-vaccinated mice. Statistical significance was determined usingone-way analysis of variance (repeated measures ANOVA) (*P<0.05;**P<0.01; ***P<0.001) with multiple comparison test (#P<0.05). Each dotrepresents the individual endpoint titer Log₁₀ of IgG of a mouse. Theaverage value of each group is represented by a horizontal line.

FIG. 49 shows the Log₁₀ 1/endpoint titer of IgG antibodies producedagainst the P. aeruginosa strain PAO1 in BALB/c mice (n=8) onpost-vaccination day 34 and in non-vaccinated control mice (withintramuscular administration). The antibody titers were determined byindirect ELISA. *P<0.0001 compared with the group of control mice;P-value according to unpaired t test. The boxes represent the first andthird quartiles; the horizontal line represents the median; the whiskersrepresent the range.

FIG. 50 is the percent survival of BALB/c mice (n=8) followingintraperitoneal infection with a 0.4× dose of P. aeruginosa PAO1 wildtype strain. Vaccinated mice were immunized on days 0, 14 and 28 with P.aeruginosa ΔPA4662 strain by intramuscular route and infected with thewild type strain at day 35. Non-vaccinated mice were administered salineon days 0, 14 and 28 and infected with the wild type strain at the sameday. *P<0.0001 survival of vaccinated group compared to non-vaccinatedgroup. P-value, according to the Mantel-Cox test (log-rank test).

FIG. 51 shows the number of P. aeruginosa colonies (Log₁₀ CFU/mL)recovered from the blood of mice (n=3/per group) inoculated with 100 μLP. aeruginosa PAO1 wild type and ΔPA4662 strains (0.4× doses) along thetime.

FIG. 52 shows the cell viability of S. aureus wild-type (WT) and doublemutant Δmurl/Δdat (ΔΔ) strains on TSB plates supplemented withD-glutamate immediately (growth control) and after 12 and 18 hours ofdrought stress at 37° C. All cultures and dilution series were performedin triplicate.

FIG. 53 is the percent survival of BALB/c mice (n=5) followingintraperitoneal infection with S. aureus 132 wild type strain. Mice werepassively immunized with vaccine serum or naïve serum. *P=0.0429survival of mice administered vaccine serum compared to mice receivednaïve serum. P-value, according to the Mantel-Cox test (log-rank test).

DESCRIPTION OF THE INVENTION Definitions

In the context of the present invention, the term “D-glutamate” isunderstood as the compound or molecule with molecular formula C₅H₉NO₄,molecular weight 147.129 (g/mol) and having the D-enantiomer form ofglutamate. Its systematic name is “D-glutamic acid”, but it can also bedesignated as (without being limited to) “D-Glu”, “D-2-amino pentanoicacid”, “glutamic acid D-form”, “(R)-2-amino pentanoic acid” andH-D-Glu-OH. Its nomenclature in the IUPAC (International Union of Pureand Applied Chemistry) system is (2R)-2-amino pentanoic acid and itsidentifier in the “PubChem Compound” database is 6893-26-1.

In the context of the present invention, the term “glutamate racemase”is understood as the protein catalyzing the interconversion reaction ofL-glutamate to D-glutamate, which is necessary for bacterial wallsynthesis. Its EC identifier (Enzyme Commission number) is 5.1.1.3. Thisprotein is easily and invariably identified in nucleotide or amino acidsequence databases by its EC code, which refers to an enzyme of whichthe catalytic activity is L-glutamate=D-glutamate, because thedesignation thereof can be variable. Therefore, in some Acinetobacterbaumannii strains, the glutamate racemase enzymatic function can beattributed to proteins the designation of which isL-alanine-LD-glutamate racemase/epimerase, Asp/Glu/hydantoin racemase,bacitracin synthetase 1 (BA1), aspartate/glutamate racemase, amongothers.

In the context of the present invention, the term “D-amino acidtransaminase” is understood as the protein catalyzing theinterconversion reaction of D-alanine and 2-oxoglutarate to pyruvate andD-glutamate. Its EC identifier is 2.6.1.21. This protein is easily andinvariably identified in nucleotide or amino acid sequence databases byits EC code, which refers to an enzyme of which the catalytic activityis D-alanine+2-oxoglutarate=pyruvate+D-glutamate, because thedesignation thereof can be variable.

In the context of the present invention, the term “auxotrophic forD-glutamate” is understood as the lack of a functional metabolic pathwaygenerating the D-glutamate substance, on which the thus designatedbacterium depends for growth, due to the inability to synthesize thiscompound.

In the context of the present invention, the term “Murl” is understoodas being synonymous with the term “glutamate racemase”.

In the context of the present invention, the term “Dat” is understood asbeing synonymous with the term “D-amino acid transaminase”.

In the context of the present invention, the term “murl” is understoodas a gene or nucleotide sequence encoding a glutamate racemase protein.Depending on the Acinetobacter baumannii, Pseudomonas aeruginosa andStaphylococcus aureus strain, the chromosomal genes encoding theglutamate racemase protein can be called (without being limited to)murl, murl_1 or murl_2, or they can be uniquely identified by theirchromosomal locus.

In the context of the present invention, the term “dat” is understood asa gene or nucleotide sequence encoding a D-amino acid transaminaseprotein. Depending on the Staphylococcus aureus strain, the chromosomalgenes encoding the D-amino acid transaminase protein can be called(without being limited to) dat, or it can be uniquely identified bytheir chromosomal locus.

In the context of the present invention, the term “inactivation” isunderstood as the blocking of the expression of a specific gene or of aprotein either through molecular modification or negative regulation ofone or both. Molecular modification includes the use of conventionalrecombinant DNA techniques which in turn include: the substitution ofone or several nucleotides, the insertion of one or several nucleotides,the partial or complete deletion of a gene, chemically-induced orradiation-induced disruption by mutagenesis. Negative regulation of theexpression of a gene or protein includes transcriptional andpost-transcriptional gene silencing.

In the context of the present invention, the term “Acinetobacterbaumannii” is defined as any microorganism belonging to the “Bacteria”domain, “Proteobacteria” phylum, “Gammaproteobacteria” class,“Pseudomonadales” order, “Moraxellaceae” family, “Acinetobacter” genus,“calcoaceticus/baumannii” complex and “A. baumannii” species.

The microorganisms thus defined are characterized by being gramnegative, strictly aerobic, non-fermenting and oxidase-negative.

In the context of the present invention, the term “ATCC 17978” refers toany bacterial strain with the identifier 17978 in the American TypeCulture Collection and belonging to the “Bacteria” domain,“Proteobacteria” phylum, “Gammaproteobacteria” class, “Pseudomonadales”order, “Moraxellaceae” family, “Acinetobacter” genus,“calcoaceticus/baumannii” complex and “A. baumannii” species.

In the context of the present invention, the term “locus A2S_0380” isdefined as physical position “0380” in the chromosome of A. baumanniistrain ATCC 17978 the gene of which is referred to as A1S_0380 or murland the product of which is glutamate racemase protein.

In the context of the present invention, the term “locus A1S_3398” isdefined as physical position “3398” in the chromosome of A. baumanniistrain ATCC 17978 the gene of which is referred to as A1S_3398 or murland the product of which is glutamate racemase protein.

In the context of the present invention, the term “Δ0380” is defined asthe absence of locus A1S 0380 in the chromosome of Acinetobacterbaumannii strain ATCC 17978.

In the context of the present invention, the term “Δ3398” is defined asthe absence of locus A1S_3398 in the chromosome of Acinetobacterbaumannii strain ATCC 17978.

In the context of the present invention, the term “double mutationΔ0380/Δ3398” is defined as the simultaneous absence of loci A1S_0380 andA1S_3398 in the chromosome of Acinetobacter baumannii strain ATCC 17978.

In the context of the present invention, the term “ATCC 19606” refers toany bacterial strain with the identifier 19606 in the American TypeCulture Collection and belonging to the “Bacteria” domain,“Proteobacteria” phylum, “Gammaproteobacteria” class, “Pseudomonadales”order, “Moraxellaceae” family, “Acinetobacter” genus,“calcoaceticus/baumannii” complex and “A. baumannii” species.

In the context of the present invention, the term “AbH12O-A2” as usedherein, refers to the bacterial strain thus designated and belonging tothe “Bacteria” domain, “Proteobacteria” phylum, “Gammaproteobacteria”class, “Pseudomonadales” order, “Moraxellaceae” family, “Acinetobacter”genus, “calcoaceticus/baumannii” complex and “A. baumannii” species. Itis a highly invasive strain which was isolated in a hospital outbreakthat caused several patients to die, and is characterized by itsresistance pattern with respect to multiple antibiotics (described inMerino et al., Antimicrob Agents Chemother, 54(6):2724-7 (2010)).

In the context of the present invention, the term “Ab307-0294”, as usedherein, refers to the bacterial strain thus designated and belonging tothe “Bacteria” domain, “Proteobacteria” phylum, “Gammaproteobacteria”class, “Pseudomonadales” order, “Moraxellaceae” family, “Acinetobacter”genus, “calcoaceticus/baumannii” complex and “A. baumannii” species.This is a highly virulent capsulated strain of A. baumannii that hasbeen studied as a model pathogen (as described in Russo et al, Infectionand Immunity, 78 (9): 3993-4000 (2010)).

In the context of the present invention, the term “Pseudomonasaeruginosa” is defined as any organism belonging to the “Bacteria”domain, “Proteobacteria” phylum, “Gammaproteobacteria” class,“Pseudomonadales” order, “Pseudomonadaceae” family, “Pseudomonas” genusand “P. aeruginosa” species. P. aeruginosa is a Gram negative, aerobic,coccobacillus bacterium with unipolar motility.

In the context of the present invention, the term “PAO1” refers to anybacterial strain with the universal identifier PAO1 and belonging to the“Bacteria” domain, “Proteobacteria” phylum, “Gammaproteobacteria” class,“Pseudomonadales” order, “Pseudomonadaceae” family, “Pseudomonas” genusand “P. aeruginosa” species.

In the context of the present invention, the term “PA4662” is defined asphysical position “4662” in the chromosome of P. aeruginosa strain PAO1the gene of which is referred to as PA4662 or murl and the product ofwhich is glutamate racemase protein.

In the context of the present invention, the term “ΔPA4662” is definedas the absence of locus PA4662 in the chromosome of Pseudomonasaeruginosa strain PAO1.

In the context of the present invention, the terms “PA28562”,“PA51430664”, “PA26132” and “PA29475”, as used herein, refer to thebacterial strains thus designated and belonging to the “Bacteria”domain, “Proteobacteria” phylum, “Gammaproteobacteria” class,“Pseudomonadales” order, “Pseudomonadaceae” family, “Pseudomonas” genusand “P. aeruginosa” species.

In the context of the present invention, the term “PAST175”, as usedherein, refers to the bacterial strain with the MLST sequence type 175(ST175) and belonging to “Bacteria” domain, “Proteobacteria” phylum,“Gammaproteobacteria” class, “Pseudomonadales” order, “Pseudomonadaceae”family, “Pseudomonas” genus and “P. aeruginosa” species. This strain waspreviously identified as an internationally spred high-risk clone, asdescribed in M. Garcia-Castillo et al, Journal of Clinical Microbiology49 (2011) 2905-2910.

In the context of the present invention, the term “PA12142”, as usedherein, refers to the bacterial strain 12142 (epidemic Liverpool strain)as described in M. Tomás et al, Antimicrobial Agents and Chemotherapy 54(2010) 2219-2224. This strain belongs to “Bacteria” domain,“Proteobacteria” phylum, “Gammaproteobacteria” class, “Pseudomonadales”order, “Pseudomonadaceae” family, “Pseudomonas” genus and “P.aeruginosa”.

In the context of the present invention, the term “Staphylococcusaureus” is defined as any microorganism belonging to the “Bacteria”domain, “Firmicutes” phylum, “Bacilli” class, “Bacillales” order,“Staphylococcaceae” family, “Staphylococcus” genus and “S. aureus”species. The microorganisms thus defined are characterized by being Grampositive, facultative anaerobic, coccal bacterium.

In the context of the present invention, the term “132” refers to anybacterial strain with the same designation and belonging to the“Bacteria” domain, “Firmicutes” phylum, “Bacilli” class, “Bacillales”order, “Staphylococcaceae” family, “Staphylococcus” genus and “S.aureus” species. This is a clinical methicillin resistant strain(Vergara-Irigaray et al, Infection and Immunity, 77 (9)3978-3991(2009)), and was used in this invention as a model to generate anauxotrophic mutant of “Staphylococcus aureus”.

In the context of the present invention, the term “Δmurl” is defined asthe absence of locus murl in the chromosome of Staphylococcus aureusstrain 132.

In the context of the present invention, the term “Δdat” is defined asthe absence of locus dat in the chromosome of Staphylococcus aureusstrain 132.

In the context of the present invention, the term “double mutationΔmurl/Δdat” is defined as the simultaneous absence of murl and dat genesin the chromosome of Staphylococcus aureus strain 132.

In the context of the present invention, the term “132 Δspa”, as usedherein, is meant a microorganism with the same designation and belongingto the “Bacteria” domain, “Firmicutes” phylum, “Bacilli” class,“Bacillales” order, “Staphylococcaceae” family, “Staphylococcus” genusand “S. aureus” species. The microorganism thus defined is a S. aureus132 strain with deletion in the spa gene (Vergara-Irigaray et al,Infection and Immunity, 77 (9): 3978-3991 (2009)).

In the context of the present invention, the term “RN4220”, as usedherein, is meant a microorganism with the same designation and belongingto the “Bacteria” domain, “Firmicutes” phylum, “Bacilli” class,“Bacillales” order, “Staphylococcaceae” family, “Staphylococcus” genusand “S. aureus” species. The microorganism thus defined is a S. aureuscloning intermediate strain (Nair et al, Journal Bacteriology, 193(9):2332-2335 (2011)).

In the context of the present invention, the term “USA300LAC”, as usedherein, is meant a microorganism with the same designation and belongingto the “Bacteria” domain, “Firmicutes” phylum, “Bacilli” class,“Bacillales” order, “Staphylococcaceae” family, “Staphylococcus” genusand “S. aureus” species. The microorganism thus defined is an epidemicmethicillin-resistant S. aureus strain responsible for communityacquired infections in healthy individuals (Diep et al, Lancet 4;397(9512):731-739 (2006)).

In the context of the present invention, the term “RF122”, as usedherein, refers to the bacterial strain with the MLST sequence type 151(ST175) and clonal complex 151 (CC151) and belonging to the “Bacteria”domain, “Firmicutes” phylum, “Bacilli” class, “Bacillales” order,“Staphylococcaceae” family, “Staphylococcus” genus and “S. aureus”species. The microorganism thus defined is a bovine mastitis-causingstrain isolated from a cow presenting clinical mastitis (Herron-Olson Let al, PloS ONE 2:e1120 (2007)).

In the context of the present invention, the term “ED133”, as usedherein, refers to the bacterial strain with the MLST sequence type 133(ST175) and clonal complex 133 (CC133) and belonging to the “Bacteria”domain, “Firmicutes” phylum, “Bacilli” class, “Bacillales” order,“Staphylococcaceae” family, “Staphylococcus” genus and “S. aureus”species. The microorganism thus defined is an ovine mastitis-causingstrain isolated in France (Guinane et al, Genome Biol Evol 2:454-466(2010)).

In the context of the present invention, the term “ED98”, as usedherein, refers to the bacterial strain with the MLST sequence type 5(ST5) and clonal complex 5 (CC5) and belonging to the “Bacteria” domain,“Firmicutes” phylum, “Bacilli” class, “Bacillales” order,“Staphylococcaceae” family, “Staphylococcus” genus and “S. aureus”species. The microorganism thus defined is an avian-adapted strainisolated from a diseased broiler chicken (Lowder et al, PNAS106(46)19545-50 (2009)).

In the context of the present invention, the term “glutamateracemase-deficient bacterial strains” is understood as any bacterialstrain unable to produce a functional and/or active form of glutamateracemase enzyme. This deficiency can be due to: blocking of theexpression of the coding genes thereof, post-transcriptionalmodifications and post-translational modifications affecting enzymaticactivity, allosteric regulation or the cellular location of this enzyme.

In the context of the present invention, the term “passive immunization”is used to refer to the administration of antibodies or fragmentsthereof to an individual with the intent of conferring immunity to thatindividual.

In the context of the present invention, the expression “therapeuticallyeffective amount” refers to the amount of antibodies of the invention orof attenuated bacterial strains of the invention that allow producingthe desired effect. The pharmaceutically acceptable adjuvants andcarriers that can be used in said compositions are carriers known bypersons skilled in the art. The compositions provided by this inventioncan be facilitated through any administration route, for which purposesaid composition will be formulated in the suitable dosage form and withthe excipients that are pharmacologically acceptable for the chosenadministration route.

In the context of the present invention, the term “vaccine” refers to anantigenic preparation used to establish an immune system response to adisease.

DETAILED DESCRIPTION OF THE INVENTION

As already stated, a bacterial strain is auxotrophic for D-glutamate ifit has completely lost the ability to produce D-glutamate. Throughoutthe present invention we shall show that bacterial strains auxotrophicfor D-glutamate have characteristics that make them especially suitablefor use as vaccines. This is the case, since these specific types ofbacterial strains (auxotrophic for D-glutamate) are sufficientlya-virulent (attenuated) to avoid unacceptable pathological effects,induce a sufficient level of immunity in the host and have substantiallyno probability for reverting to a virulent wild type strain.

All of these characteristics provide for a novel technological platformfor the design and production of vaccines having potential applicationin a wide variety of bacterial strains (universality). This fact (theversatility or universality of the invention) is clearly demonstratedthroughout the present invention since the authors have put theinvention into practice in three completely unrelated bacterial species(Acinetobacter baumannii (A. baumannii), Pseudomonas aeruginosa (P.aeruginosa) and Staphylococcus aureus (S. aureus)). For each of thesecompletely unrelated bacterial species D-glutamate auxotrophic strainswere produced and as demonstrated in the examples below, all of thesemutant strains were sufficiently a-virulent (attenuated) to avoidunacceptable pathological effects but capable of inducing a sufficientlevel of immunity in the host. Consequently all of these strains areexcellent vaccine candidates.

In order to demonstrate the above, the authors of the present inventionfirst needed to prove that all auxotrophic D-glutamate strains gave riseto attenuated strains incapable of producing unacceptable pathologicaleffects in the host. For this purpose the authors used nosocomialpathogen A. baumannii (see example 5).

For this purpose and as shown in example 5, the authors of the presentinvention induced a systemic infection in BALB/c mice through, on theone hand, the intraperitoneal administration of an inoculum of A.baumannii wild type strain ATCC 17978 and through, on the other hand,the intraperitoneal administration of an inoculum of the double mutantstrain Δ0380/Δ3398 (an A. baumannii strain deficient in the enzymeglutamate racemase and thus a D-glutamate auxotrophic strain).

FIG. 10A illustrates part of the results from this experiment; inparticular the different percentages of survival of mice when infectedwith increasing doses of the wild type strain. From this data it can beconcluded that doses from 2× onwards provide for a gradual decrease inthe survival of the mice. In fact, it can be observed that the lethaldose 100 (LD₁₀₀), understood as the minimum dose necessary to reducesurvival of mice to 0%, for wild type strain ATCC 17978 is 2.5×.Moreover, this figure also shown that when the dose is above 3×, a muchrapid reduction in the survival percentage is observed.

On the other hand, FIG. 10B shows different levels of survival rates ofmice infected with increasing doses of the double mutant strainΔ0380/Δ3398. In this case and in clear contrast with FIG. 10A, the LD₁₀₀(lethal dose 100) is 6×. This dose is certainly much higher than thelethal dose corresponding to the wild type strain. In fact, a 6× dose isso high that the authors of the invention firmly believe that the deathof the mice was not due to the replication of the bacteria but to aseptic shock, which clearly indicates that this (the double mutantstrain) is an a-virulent or attenuated bacterial strain.

Additionally and in order to confirm the above results, the authors ofthe present invention induced, as can be observed in example 6, asystemic infection in BALBIc mice through the intraperitonealadministration of the following inoculums of A. baumannii: wild typestrain ATCC 17978 and mutant strains Δ0380, Δ3398 and Δ0380/Δ3398.

In this sense, it is important to note that as illustrated in theexamples of the present invention, of all of the strains used andmentioned in the precedent paragraph, only the double mutant strainΔ0380/Δ3398 is considered to be auxotrophic for D-glutamate thusrequiring the addition of exogenous D-glutamate in the culture mediumfor growth and survival. In addition, it is also noted that in the modelof acute infection illustrated in example 6, the infection occurs with arapid spread through the blood of the bacteria thus resulting in thedeath of the mice between 11 and 30 hours post-infection (see FIG. 10A).Lastly, it is also important to note that from the counts of bacteria inthe liver, the authors of the present invention can obtain a measurementof the invasive and replicative capacity of the different strains.

That said and as shown in FIG. 11, the following average values wereobtained from the experiment illustrated in example 6: 8.29 Log₁₀ CFU(colony forming units)/g in the liver of those mice infected with thewild type strain; 6.88 Log₁₀ CFU/g in the liver of those mice infectedwith the Δ0380 strain; 8.06 Log₁₀ CFU/g in the liver of those miceinfected with the mutant strain Δ3398 and 1.59 Log₁₀ CFU/g in the liverof those mice infected with the double mutant strain Δ0380/Δ3398.

Based on these results, it can be concluded that the counts of thedouble mutant strain Δ0380/Δ3398 and of mutant strain Δ0380 wereconsiderably inferior from those observed for the wild type strain. Themost dramatic and obvious reduction being the one observed whenanalyzing the colony counts of the double mutant strain Δ0380/Δ3398, inwhich a decrease of nearly 7 Log₁₀ values in the average bacterial loadwas obtained. Moreover, surprisingly in 44.4% of mice infected with thedouble mutant strain no bacteria were recovered.

Consequently, again this clearly indicates that this (the double mutantstrain) is an a-virulent or attenuated bacterial strain.

Nevertheless, the authors of the present invention in order to confirmthe universality of the platform, conducted further experiments withauxotrophic D-glutamate strains pertaining to two additional andcompletely unrelated bacterial species, namely with P. aeruginosa and S.aureus. As we shall see below, auxotrophic strains of these two speciesalso produced attenuated strains incapable of producing unacceptablepathological effects in the host.

In this sense, as illustrated in example 16, BALB/c mice wereadministered different doses of P. aeruginosa PAO1 wild type strain andof mutant strain ΔPA4662 with the purpose of determining the lethaldoses of these strains during an acute sepsis infection. Mice weremonitored for 7 days after infection and survival rates were determinedfor different doses of injected bacteria.

In FIG. 24A we can observe different degrees of survival in animalsinfected with increasing doses of P. aeruginosa PAO1 wild type strain.For this strain, the LD₁₀₀ is =0.4×. In FIG. 24B we can observedifferent degrees of survival in animals infected with increasing dosesof the ΔPA4662 mutant strain. For this strain, the LD₁₀₀ is >40×, a veryhigh dose of bacterial inoculum which can lead to the death of the micefrom septic shock (and not due to replication of the bacteria). Thisindicates that this strain (ΔPA4662) has a much reduced virulence incomparison with the wild type strain (a dose 100 times higher than thewild type strain LD₁₀₀ only decreases by 50% the survival rate of themice).

Lastly the authors of the present invention further confirmed theconcept of universality of the platform technology of the invention byusing a still further bacterial species, namely by proving thatauxotrophic D-glutamate strains pertaining to the species S. aureus alsoproduced attenuated strains incapable of producing unacceptablepathological effects in the host.

In this sense and as illustrated in example 25, systemic infection wasproduced in BALB/c mice with S. aureus 132 wild type and double mutantstrains by intraperitoneal injection with 3% of hog mucin.

As shown in FIG. 35A, the minimum dose of the wild type strain thatreduces survival of these mice to 0% was determined to be LD₁₀₀=3×. Inclear contrast with FIG. 35A, FIG. 35B illustrates that inoculating adose of the double mutant 10-fold higher than the LD₁₀₀ of the wild typestrain results in a 100% survival rate. Therefore, the lethal dose forthe double mutant is greater than 30× LD₁₀₀>30×. This clearlydemonstrates that the double mutant of S. aureus is an attenuated strainshowing lower virulence potential than the wild type counterpart.

In summary these data demonstrate that all bacterial strains auxotrophicfor D-glutamate are attenuated. However, it is widely known that it isnot enough that a bacterial strain is attenuated to be useful as avaccine candidate since it must also be able to generate an immuneresponse and induce protection. Furthermore, these mutant strains shouldpreferentially contain all antigens immunologically necessary to confercross-protection.

Thus, in order to assess the immune response mediated by antibodies todifferent vaccination regimens, the authors conducted the experimentillustrated in example 8 wherein BALB/c mice were immunized byintraperitoneal injection with the double mutant strain Δ0380/Δ3398 byusing a 1× dose on days 0 and 14. Additionally, a further group ofcontrol mice were administered of saline serum identically at days 0 and14. On the seventh day post-immunization, sera from the mice immunizedwith a single dose of the vaccine (administered on day 0) was retrieved,similarly, 21 days after the first administration, sera from the micealso immunized with the remaining dose was also retrieved along with thesera from the control mice, from those injected with saline serum.

These sera were used to determine the titer of antibodies (IgG) by usingthe ELISA technique against different strains of A. baumannii, includingATCC 17978, ATCC 19606 and AbH12O-A2 and thus measuring the ability ofthe vaccine to generate broad immune response (example 9). It isnoteworthy that A. baumannii strain AbH12O-A2 is a highly invasivestrain, isolated in a hospital outbreak that killed several patients andis characterized by a pattern of resistance to multiple antibiotics.

In all immunized mice significant levels of antibodies were detectedagainst the wild type strain as compared to non-vaccinated mice (FIG.13). Antibody production was significantly elevated at days 7 and 21,namely after injection of the first dose and of the second dose of thedouble mutant bacterial strain Δ0380/Δ3398, respectively as compared tonon-vaccinated mice. Moreover, the production of antibodies at day 21(after 2 successive injections of the double mutant strain Δ0380/Δ3398)was significantly higher than the production of antibodies at day 7. Theproduction of antibodies obtained at day 21 against A. baumannii wildtype strain ATCC 17978 were similar to those obtained against A.baumannii strains ATCC 19606 and A. baumannii AbH12O-A2 (FIG. 15). Thisresult demonstrates that immunization with strain Δ0380/Δ3398 not onlygenerates antibodies against the wild type strain but also generates IgGantibodies that react against other bacterial strains with differentresistance and virulence patterns such as the ATCC 19606 strain andstrain AbH12O-A2.

In addition, to further confirm that these bacteria (bacterial strainsauxotrophic for D-glutamate) were capable of generating a strong immuneresponse, further experiments were conducted in bacterial species P.aeruginosa and S. aureus.

As illustrated in example 16, one vaccine dose of 0.1× of P. aeruginosaΔPA4662 mutant strain is sufficient to trigger IgG productionsignificantly, even when detected at day 40^(th) after theadministration (FIG. 44). Nonetheless, vaccine doses equal or greaterthan 0.4× elicit higher levels of IgG production. Moreover, IgGproduction is significantly higher when the 2^(nd) vaccine dose isadministered (FIG. 45).

Additionally, ELISA was performed with respect to different strains ofP. aeruginosa with sera obtained from mice vaccinated with the ΔPA4662and mice administrated saline to measure the capacity of ΔPA4662 vaccineto generate a broad immune response. Results similar to those observedwith respect to P. aeruginosa strain PAO1 were obtained with strainPA28562 whereas high levels of cross-reactivity were also seen withrespect to rest of P. aeruginosa strains tested (FIG. 46). Thisdemonstrates that immunization with strain ΔPA4662 not only generatesantibodies against the isogenic wild type strain, but also generates lgGantibodies that react against multiple P. aeruginosa strains.

Lastly, as illustrated in examples 28 and 29 the authors of the presentinvention evaluated the immune response mediated by antibodies against astrain of S. aureus (called S. aureus 132 Δspa, Protein-A-deficient) andthe cross-reactivity against four unrelated S. aureus strains of humanand animal origin. For this purpose, BALB/c mice were immunized byintraperitoneal injection of the double mutant Δmurl/Δdat on days 0 and14. The sera samples from each mouse collected on day 21 were used todetermine the titer of antibodies (IgG) against the isogenic S. aureus132 Δspa strain (FIG. 39), as well as against an epidemic MRSAcommunity-adquired USA300LAC strain and three strains of animal origin(FIG. 40). As shown in FIGS. 39 and 40, all mice immunized with thedouble mutant Δmurl/Δdat strain produced significant levels of specificantibodies against each bacteria as compared with the non-vaccinatedmice, demonstrating the ability of the S. aureus Δmurl/Δdat mutant toelicit IgGs antibodies that recognize not only the isogenic Δspacounterpart but also a clinical epidemic strain and other strainsisolated from different hosts such as bovine, ovine and poultry.

Therefore, the data presented so far not only demonstrates that allbacterial strains auxotrophic for D-glutamate are attenuated but alsothat these strains contain all antigens immunologically necessary togenerate an immune response and to confer cross-protection.

Finally, to verify whether the bacterial strains auxotrophic forD-glutamate confer an acceptable level of protection, the authors of thepresent invention conducted a series of experiments with nosocomialpathogen A. baumannii in order to assessed the effectiveness as avaccine of the double mutant strain Δ0380/Δ3398. As illustrated inexample 10, mutant strain Δ0380/Δ3398 was administered to BALB/c mice ondays 0 and 14. Control mice were administered only saline identically atdays 0 and 14. Twenty one days after the first injection, mice werechallenged with A. baumannii strains ATCC 17978, AbH12O-A2 andAb307-0294, independently, in order to establish a lethal systemicinfection. After the challenge, mice were monitored for 7 days todetermine the survival rate of vaccinated mice compared to control mice(non-vaccinated).

When infected with A. baumannii ATCC 17978 strain, 11 deaths wereobserved in the group of unvaccinated mice during the first 24 hours,which means a mortality rate of 92% in this group. In contrast, allvaccinated mice survived to the challenge, overcoming the infection,which means a 100% survival (see FIG. 16) rate in this group.

In addition, it was determined whether the response produced byimmunization with the Δ0380/Δ3398 strain was sufficient to provideprotection from lethal infection with other A. baumannii strains,including highly virulent and pathogenic strains. In the case ofchallenge with the AbH12O-A2 strain, 9 deaths were observed in the groupof non-vaccinated mice during the first 19 hours, which means amortality rate of 100%. In contrast, all vaccinated mice survived (100%survival rate) (see FIG. 17).

In the case of challenge with the Ab307-0294 capsulated strain, werecorded a 100% mortality rate in the group of non-vaccinated micewithin the first 24 hours and a 83% survival rate in the group of micepreviously immunized with the Δ0380/Δ3398 strain (see FIG. 18). Thisconfirms that vaccination with the mutant strain confers protectionagainst a systemic infection caused by an A. baumannii strain withmarked virulence.

All these results suggest that vaccination with the Δ0380/Δ3398 straincan provide protective immunity against infection with a diverse groupof A. baumannii strains.

Furthermore, in order to verify whether other bacterial strains alsoauxotrophic for D-glutamate confer an acceptable level of protection,the authors of the present invention conducted additional experimentswith pathogens P. aeruginosa (as illustrated in example 18) and S.aureus (as illustrated in examples 26 and 27).

As illustrated in example 18, mutant strain PA4662 was administered toBALB/c mice on days 0 and 14. Control mice were administered only salineidentically days. Twenty five days after the first injection, mice werechallenged with P. aeruginosa strain PAO1, in order to establish alethal systemic infection. After the challenge, mice were monitored for7 days to determine the survival rate of vaccinated mice compared tocontrol mice.

When infected with P. aeruginosa PAO1 wild type strain, 8 deaths wereobserved in the group of non-vaccinated mice during the first 15 hours,which means a mortality rate of 100% in this group. In contrast, allvaccinated mice survived to the challenge, overcoming the infection,which means a 100% survival (see FIG. 26) rate in this group.

As illustrated in example 26, to evaluate the effectiveness (protectionlevel) of the S. aureus double mutant Δmurl/Δdat strain as a vaccine,BALB/c mice were immunized intraperitoneally with the double mutantΔmurl/Δdat on days 0 and 14. One group of mice were administered salineat identically days. At day 21, mice were infected with a lethalinoculum of S. aureus wild type strain. At 20 or 22 hours post-infection(FIGS. 36 and 37, respectively) bacterial counts in spleens and blood ofmice were determined. So, the protective effect of vaccination with thedouble mutant Δmurl/Δdat of S. aureus was confirmed as pre-immunizationwith this strain caused a significant decrease in bacterial loads.

Moreover, as indicated in example 27, when pre-immunized BALB/c micewith Δmurl/Δdat strain were challenged with a lethal dose of S. aureus132 wild type strain significant differences in survival were observedwhen compared to non-immunized mice. In this case, 8 mice of vaccinatedgroup survived to the challenge, overcoming the infection, which means a61.5% survival rate in this group (FIG. 38) whereas a mortality rate of90% were observed in non-vaccinated group.

All together, these results demostrate that vaccination with ΔPA4662 andΔmurl/Δdat s strains provides protective immunity against infection withP. aeruginosa and S. aureus, repectively.

The experimental examples described herein provide procedures andresults which establish that bacterial strains auxotrophic forD-glutamate are sufficiently a-virulent (attenuated) to avoidunacceptable pathological effects, induce a sufficient level of immunityin the host independently of the administration route and have asubstantially level of security (both enviromental and for the host) fortheir use in active or passive immunization.

Therefore, one embodiment of the invention relates to live attenuatedbacteria suitable as vaccine candidates that are no longer capable ofproducing D-glutamate.

Live attenuated bacteria for use according to the invention as vaccinecandidates can be obtained in several ways as explained below. In thissense, it is important to understand that both gram positive and gramnegative bacteria have a peptidoglycan cell wall that gives them theircharacteristic shape and provides them with mechanical protection.Peptidoglycan, also known as murein, is a polymer consisting of sugarsand amino acids that form a mesh-like layer outside the plasma membraneof bacteria (but not Archaea), forming the cell wall. The sugarcomponent consists of alternating residues of β-(1,4) linkedN-acetylglucosamine and N-acetylmuramic acid.

The peptidoglycan layer is substantially thicker in gram positivebacteria (20 to 80 nanometers) than in gram negative bacteria (7 to 8nanometers), with the attachment of the S-layer. Peptidoglycan formsaround 90% of the dry weight of gram positive bacteria but only 10% ofgram negative strains. Thus, presence of high levels of peptidoglycan isthe primary determinant of the characterization of bacteria as grampositive. In gram positive strains, it is important in attachment rolesand serotyping purposes. For both gram positive and gram negativebacteria, particles of approximately 2 nm can pass through thepeptidoglycan cell wall.

As clearly illustrated in FIGS. 1 and 2, D-glutamate is one of the maincomponents of the peptidoglycan cell wall and thus D-glutamate isnecessary for cell wall peptidoglycan synthesis. Two enzymes catalyzethe formation of D-glutamate (see FIG. 3):

-   -   1. the glutamate racemase (EC 5.1.1.3), Murl, an enzyme that        catalyzes the chemical reaction        -   L-glutamate            D-glutamate        -   Thus, this enzyme has one substrate, L-glutamate, and one            product, D-glutamate; and    -   2. D-amino acid transaminase, Dat (EC 2.6.1.21), an enzyme that        catalyzes the following chemical reaction:        -   D-alanine+2-oxoglutarate            pyruvate+D-glutamate        -   Hence, the two substrates of this enzyme are D-alanine and            2-oxoglutarate, whereas its two products are pyruvate and            D-glutamate.

D-glutamate, once synthesized, is added as a monomer unit to thepeptidoglycan cell wall by an specific enzyme, namely by enzymeUDP-N-acetylmuramoyl-L-alanine: D-glutamate ligase (EC 6.3.2.9).

Therefore, the two enzymes that are capable of catalyzing the formationof D-glutamate are glutamate racemase and D-amino acid transaminase.However, the enzyme D-amino acid transaminase is not present in allbacterial strains. In contrast, the glutamate racemase gene is conservedin all species that produce peptidoglycan as Acinetobacter baumannii,Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Enterococcusfaecalis, Enterococcus faecium, Escherichia coli, Helicobacter pylori,Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa,Streptococcus pyogenes, etc.

In many of these bacteria, including E. coli, there is thus a soleprotein capable of catalyzing the synthesis of D-glutamate, namelyglutamate racemase. In these cases, this enzyme is essential forbacterial growth, since it is the sole source of D-glutamate in thecell. However, in other bacteria such as Staphylococcus haemolyticus,Bacillus sphaericus, Bacillus sp. YM -1 and S. aureus both Dat and Murlproteins are present and can thus functionally complement each other.

A. baumannii ATCC 17978 as E. coli, fails to have the Dat protein, butcomprises two different genes encoding for Murl, in particular Murl 1(locus A1S_0380) and Murl 2 (A1S_3398 locus). Both genes show a 29.8%identity between each other at the amino acid level, and 33.2 and 23.2%identity with E. coli K12 Murl protein respectively.

Therefore, an auxotrophic strain for D-glutamate can be easily obtainedthrough the inactivation of the gene or of the different genes encodingglutamate racemase Murl and also, for those bacteria further comprisingboth Dat and Murl proteins, the additional inactivation of the gene orgenes encoding D-amino acid transaminase. This means that for thosebacteria not comprising the Dat enzyme, in some cases, in order toproduce an auxotrophic strain for D-glutamate, the inactivation of asingle gene of glutamate racemase is needed, such as in the case of E.coli strain K12 and P. aeruginosa strain PAO1, in other cases theinactivation of two different genes encoding for glutamate racemase isneeded, as in the case of the strain of A. baumannii ATCC 17978, or insome other cases the inactivation of three different genes for glutamateracemase is needed, such as in the case of A. baumannii strain ABNIH10,that is, by inactivating the totality of the genes encoding glutamateracemase. In other cases, both the inactivation of the genes encodingthe protein Murl as well as those genes encoding the enzyme D-amino acidtransaminase, Dat, is required, as is the case of some gram positivebacteria like Staphylococcus aureus, Staphylococcus haemolyticus,Bacillus sphaericus and Bacillus sp. YM -1.

Consequently, a first aspect of the invention refers to a novel platformtechnology for the design and production of vaccines based on liveattenuated bacterial strains auxotrophic for D-glutamate. This novelplatform technology has potential in a wide variety of bacterial strains(universality). This fact (the versatility or universality of theinvention) has been clearly demonstrated throughout the presentinvention since the authors have putted the invention into practice inthree completely unrelated bacterial species (Acinetobacter baumannii(A. baumannii), Pseudomonas aeruginosa (P. aeruginosa) andStaphylococcus aureus (S. aureus)) as shown in the examples.

Thus, a preferred embodiment of the first aspect of the invention refersto a method for the production of a pharmaceutical composition,preferably a vaccine, comprising mutant live auxotrophic bacterialstrains for D-glutamate, wherein the pharmaceutical composition issuitable for the prophylactic treatment (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacterial strain of the composition, and wherein saidpharmaceutical composition is produced by a method comprising the stepsof:

-   -   a. obtaining mutant live auxotrophic bacterial strains for        D-glutamate;    -   b. introducing said mutant live auxotrophic bacerial strains in        a pharmaceutically acceptable carrier or diluent and optionally        adding an adjuvant; and    -   c. Optionally freeze-drying the pharmaceutical composition.

In another preferred embodiment of the first aspect of the invention,the production method comprises the steps of:

-   -   a. providing a bacterial strain capable of expressing glutamate        racemase and possibly D-amino acid transaminase and comprising a        peptidoglycan cell wall;    -   b. inactivating the gene or genes encoding for the glutamate        racemase enzyme and, if needed, the gene or genes encoding for        the enzyme D-amino acid transaminase in such way that the        bacterial strain is no longer capable of expressing a functional        glutamate racemase and/or a functional D-amino acid        transaminase, wherein the inactivation of said genes thus causes        said bacterial strain to be auxotrophic for D-glutamate; and    -   c. introducing said mutant live auxotrophic bacterial strains in        a pharmaceutically acceptable carrier or diluent and optionally        adding an adjuvant; and    -   d. Optionally freeze-drying the pharmaceutical composition.

The inactivation mentioned in step b) above can be an insertion, adeletion, a substitution or a combination thereof, provided that theinactivation leads to the failure to express a functional glutamateracemase and/or a functional D-amino acid transaminase protein. Afunctional glutamate racemase and/or a functional D-amino acidtransaminase protein is understood to be a protein having the regulatingcharacteristics of the wild-type protein. Therefore, a glutamateracemase and/or a functional D-amino acid transaminase protein that isdefective and thus incapable of participating in the synthesis ofD-glutamate is considered to be a non-functional protein.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the pharmaceutical composition is avaccine and the production method comprises adding an adjuvant.

In another preferred embodiment of the first aspect of the invention orof any of its preferred embodiments, the bacterial strain of step a) isa gram positive or gram negative bacteria. Preferably, the bacterialstrain of step a) is selected from the list of bacterial speciesconsisting of: Acinetobacter baumannii, Acinetobacter baylyi,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacterjunii, Acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacterpittii, Acinetobacter radioresistens, Actinobacillus lignieresii,Actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonasveronii subsp. sobria, Aggregatibacter actinomycetemcomitans, Arcobacterbutzleri, Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillusanthracis, Bacillus bataviensis, Bacillus cellulosilyticus, Bacilluscereus, Bacillus clausii, Bacillus licheniformis, Bacillus megaterium,Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtherias, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilla,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimills, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis. More preferably, said bacterialstrain of step a) is selected from the list consisting of the followingspecies: Acinetobacter baumannii, Pseudomonas aeruginosa andStaphylococcus aureus. Still more preferably, the bacterial strain isthe bacterial strain of A. baumannii designated Acinetobacter baumanniiDelta0380/Delta3398 and deposited under the Budapest treaty before theSpanish Type Culture Collection on Apr. 14, 2014 with strain number 8588by Fundación Profesor Novoa Santos. Still more preferably, the bacterialstrain is the bacterial strain of P. aeruginosa designated Pseudomonasaeruginosa DeltaPA4662 and deposited under the Budapest treaty beforethe Spanish Type Culture Collection on Apr. 14, 2014 with strain number8589 by FundaciOn Profesor Novoa Santos. Still more preferably, thebacterial strain is the bacterial strain of S. aureus designated132deltamurl/deltadat and deposited under the Budapest treaty before theSpanish Type Culture Collection on Jun. 11, 2014 with strain number 8587by Fundación Profesor Novoa Santos.

In addition, one further embodiment of the first aspect of the presentinventions refers to a method for the production of live attenuatedbacterial strains (from hereinafter “method for the production of liveattenuated bacterial strains of the invention”) suitable as vaccinecandidates comprising the steps of:

-   -   a. providing a bacterial strain capable of expressing glutamate        racemase and possibly D-amino acid transaminase and comprising a        peptidoglycan cell wall; and    -   b. inactivating the gene or genes encoding for the glutamate        racemase enzyme and, if needed, the gene or genes encoding for        the enzyme D-amino acid transaminase in such way that the        bacterial strain is no longer capable of expressing a functional        glutamate racemase and/or a functional D-amino acid transaminase        wherein the inactivation of said genes thus causes said        bacterial strain to be auxotrophic for D-glutamate.

In a preferred embodiment of the “method for the production of liveattenuated bacterial strains of the invention”, the bacterial strain ofstep a) is a gram positive or gram negative bacteria.

In another preferred embodiment of the “method for the production oflive attenuated bacterial strains of the invention”, the bacterialstrain of step a) has as the only way of synthesis of D-glutamate theglutamate racemase enzyme the method thus comprising the inactivation ofthe genes encoding for this enzyme, namely for glutamate racemase.

In a more preferred embodiment of the “method for the production of liveattenuated bacterial strains of the invention”, the bacterial strain ofstep a) is selected from the list of bacterial species consisting of:Acinetobacter baumannii, Acinetobacter baylyi, Acinetobactercalcoaceticus, Acinetobacter haemolyticus, Acinetobacter junkAcinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacter pittii,Acinetobacter radioresistens, Actinobacillus lignieresk Actinobacillussuis, Aeromonas caviae, Aeromonas hydrophila, Aeromonas veronii subsp.sobria, Aggregatibacter actinomycetemcomitans, Arcobacter butzleri,Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillus anthracis,Bacillus bataviensis, Bacillus cellulosilyticus, Bacillus cereus,Bacillus clausii, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacfflus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscessus, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.

In an even more preferred embodiment of the “method for the productionof live attenuated bacterial strains of the invention” said bacterialstrain of step a) is selected from the list consisting of the followingspecies: Acinetobacter baumannii, Pseudomonas aeruginosa andStaphylococcus aureus.

In a still more preferred embodiment of the “method for the productionof live attenuated bacterial strains of the invention”, said bacterialstrain of step a) is the bacterial strain of A. baumannii designatedATCC 17978 and the method comprises the inactivation of the locusA1S_0380 and A1S_3398 (see example 2).

In a still more preferred embodiment of the “method for the productionof live attenuated bacterial strains of the invention”, said bacterialstrain of step a) is the bacterial strain of P. aeruginosa designatedPAO1 and the method comprises inactivating the PA4662 gene (see example15).

In a still more preferred embodiment of the “method for the productionof live attenuated bacterial strains of the invention”, said bacterialstrain of step a) is the bacterial strain of S. aureus designated 132and the method comprises inactivating the murl and dat genes (seeexample 22).

In addition and as already stated throughout the text, because of theirunexpected attenuated but immunogenic character in vivo, the bacterialstrains as defined in the present invention are very suitable as a basisfor live attenuated vaccines.

In relation to the use as a vaccine of the bacterial strains of theinvention mentioned in the precedent paragraph, the present inventionfurther relates, namely as a second aspect of the invention, to liveattenuated pharmaceutical compositions, in particular to live attenuatedvaccine compositions, comprising the mutant auxotrophic bacterialstrains as defined herein.

These compositions are especially suitable for the protection of animalsand humans against infection with the wild type form of the mutantauxotrophic bacteria. Such animals can be selected from the groupconsisting of placental (including humans), marsupial and monotremes.Such pharmaceutical compositions, in particular vaccine compositions,comprise an immunogenically effective amount of the live attenuatedbacterium as defined herein. In addition to an immunogenically effectiveamount of the live attenuated bacterium described above, apharmaceutical composition, in particular a vaccine, according to thepresent invention also contains a pharmaceutically acceptable carrier.Such a carrier may be as simple as water, but it may e.g. also compriseculture fluid in which the bacteria were cultured. Another suitablecarrier is e.g. a solution of physiological salt concentration.

The useful dosage to be administered will vary depending on the age,weight and animal vaccinated the mode of administration and the type ofpathogen against which vaccination is sought.

The pharmaceutical composition, in particular the vaccine, may compriseany dose of bacteria, sufficient to evoke an immune response. Dosesranging between 10³ and 10¹⁰ bacteria are e.g. very suitable doses.

Optionally, one or more compounds having adjuvant activity may be addedto the pharmaceutical composition, in particular to the vaccine.Adjuvants are non-specific stimulators of the immune system. Theyenhance the immune response of the host to the vaccine. Examples ofadjuvants known in the art are Freunds Complete and Incomplete adjuvant,vitamin E, non-ionic block polymers, muramyldipeptides, ISCOMs (immunestimulating complexes, cf. for instance European Patent EP 109942),Saponins, mineral oil, vegetable oil, and Carbopol. Adjuvants,especially suitable for mucosal application are e.g. the E. coliheat-labile toxin (LT) or Cholera toxin (CT).

Other suitable adjuvants are for example aluminium hydroxide, aluminiumphosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F® or Marcol52®, saponins or vitamin-E solubilisate.

Other examples of pharmaceutically acceptable carriers or diluentsuseful in the present invention include stabilisers such as SPGA,carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose,dextran), proteins such as albumin or casein, protein containing agentssuch as bovine serum or skimmed milk and buffers (e.g. phosphatebuffer). Especially when such stabilisers are added to the vaccine, thevaccine is very suitable for freeze-drying. Therefore, in a morepreferred form, the vaccine is in a freeze-dried form.

For administration to animals or humans, the pharmaceutical composition,in particular the vaccine, according to the present invention can begiven inter alia intranasally, intradermally, subcutaneously, orally, byaerosol or intramuscularly. For application to poultry, wing web andeye-drop administration are very suitable. The medicament,pharmaceutical composition or vaccine of the invention can be used bothin asymptomatic patients as well as in those who have already shownsymptoms of the disease.

Therefore, a second aspect of the invention refers to a pharmaceuticalcomposition, preferably a vaccine, comprising mutant live auxotrophicbacerial strains for D-glutamate and a pharmaceutically acceptablecarrier or diluent and optionally an adjuvant, wherein saidpharmaceutical composition is suitable for the prophylactic (beforeinfection) and/or therapeutic treatment (after infection or after theclinical manifestation of the disease caused by the infection) ofanimals and/or humans against infection with the wild type form of themutant auxotrophic bacteriaof the composition.

In a preferred embodiment of the second aspect of the invention, saidpharmaceutical composition is a vaccine and said vaccine optionallycomprises an adjuvant.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutically acceptablecarrier or diluent is selected from the list consisting of water,culture fluid, a solution of physiological salt concentration and/orstabilisers such as SPGA, carbohydrates (e.g. sorbitol, mannitol,starch, sucrose, glucose, dextran), proteins such as albumin or casein,protein containing agents such as bovine serum or skimmed milk andbuffers (e.g. phosphate buffer).

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said adjuvant is selected from thelist consisting of Freunds Complete and Incomplete adjuvant, vitamin E,non-ionic block polymers, muramyldipeptides, ISCOMs (immune stimulatingcomplexes), Saponins, mineral oil, vegetable oil, Carbopol, the E. coliheat-labile toxin (LT) or Cholera toxin (CT), aluminium hydroxide,aluminium phosphate or aluminium oxide, oil-emulsions (e.g. of Bayol F®or Marcol 52®, saponins and vitamin-E solubilisate.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutical compositioncomprises a dose of mutant live auxotrophic bacterial strains forD-glutamate ranging between 10³ and10¹⁰ bacteria.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, said pharmaceutical composition isin a freeze-dried form.

In another preferred embodiment of the second aspect of the invention orof any of its preferred embodiments, the bacterial strain is selectedfrom the list of bacterial species consisting of: Acinetobacterbaumannii, Acinetobacter baylyi, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Acinetobacter junk Acinetobacter lwoffii,Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacterradioresistens, Actinobacillus lignieresii, Actinobacillus suis,Aeromonas ca viae, Aeromonas hydrophila, Aeromonas veronii subsp.sobria, Aggregatibacter actinomycetemcomitans, Arcobacter butzleri,Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillus anthracis,Bacillus bataviensis, Bacillus cellulosilyticus, Bacillus cereus,Bacillus clausii, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illni, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorefta asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.

More preferably, said bacterial strain of step a) is selected from thelist consisting of the following species: Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus. Still more preferably,the bacterial strain is the bacterial strain of A. baumannii designatedAcinetobacter baumannii Delta0380/Delta3398 and deposited under theBudapest treaty before the Spanish Type Culture Collection on Apr. 14,2014 with strain number 8588 by Fundacion Profesor Novoa Santos. Stillmore preferably, the bacterial strain is the bacterial strain of P.aeruginosa designated Pseudomonas aeruginosa DeltaPA4662 and depositedunder the Budapest treaty before the Spanish Type Culture Collection onApr. 14, 2014 with strain number 8589 by Fundación Profesor NovoaSantos. Still more preferably, the bacterial strain is the bacterialstrain of S. aureus designated 132deltamurl/deltadat and deposited underthe Budapest treaty before the Spanish Type Culture Collection on Jun.11, 2014 with strain number 8587 by Fundación Profesor Novoa Santos.

A third aspect of the invention refers to a live attenuated bacterialstrain, obtain or obtainable by means of the method of the first aspectof the invention or by means of any of the preferred embodiments of thefirst aspect of the invention.

A different embodiment of the third aspect of the present inventionrelates to a live attenuated D-glutamate auxotrophic bacterial strain,suitable as a vaccine, characterized in that in said strain the genesencoding for the glutamate racemase enzyme and, if existent, the genesencoding for the enzyme D-amino acid transaminase, are inactivated.

Such inactivation can be an insertion, a deletion, a substitution or acombination thereof, provided that the inactivation leads to the failureto express a functional glutamate racemase and a functional D-amino acidtransaminase protein. A functional glutamate racemase and/or afunctional D-amino acid transaminase protein is understood to be aprotein having the regulating characteristics of the wild-type protein.Therefore, a glutamate racemase and/or a functional D-amino acidtransaminase protein that is defective and thus incapable ofparticipating in the synthesis of D-glutamate is considered to be anon-functional protein.

In a preferred embodiment of the third aspect of the invention, thebacterial strain is a gram positive or gram negative bacteria.

In another preferred embodiment of the third aspect of the invention,the bacterial strain has as the only way of synthesis of D-glutamate theglutamate racemase enzyme the bacteria thus being characterized by theinactivation of the genes encoding for this enzyme, namely for glutamateracemase.

In a more preferred embodiment of the third aspect of the invention, thebacterial strain is selected from the list of bacterial speciesconsisting of: Acinetobacter baumannii, Acinetobacter baylyi,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacterjunii, Acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacterpittii, Acinetobacter radioresistens, Actinobacillus lignieresii,Actinobacillus suis, Aeromonas caviae, Aeromonas hydrophile, Aeromonasveronii subsp. sobria, Aggregatibacter actinomycetemcomitans, Arcobacterbutzleri, Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillusanthracis, Bacillus bataviensis, Bacillus cellulosilyticus, Bacilluscereus, Bacillus clausii, Bacillus licheniformis, Bacillus megaterium,Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragallinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscesses, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veilloneliadispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.

In an even more preferred embodiment of the third aspect of theinvention said bacterial strain is selected from the list consisting ofthe following species: Acinetobacter baumannii, Pseudomonas aeruginosaand Staphylococcus aureus.

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is the bacterial strain of A. baumanniidesignated ATCC 17978 characterized by the inactivation of the locusA1S_0380 and A1S_3398 (see example 2).

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is the bacterial strain of P.aeruginosa designated PAO1 characterized by the inactivation of thePA4662 gene (see example 15).

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is a bacterial strain of S. aureusdesignated 132 characterized by the inactivation of the murl and datgenes (see example 22).

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is the bacterial strain of A. baumanniidesignated Acinetobacter baumannii Delta0380/Delta3398 and depositedunder the Budapest treaty before the Spanish Type Culture Collection onApr. 14, 2014 with strain number 8588 by Fundación Profesor NovoaSantos.

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is the bacterial strain of P.aeruginosa designated Pseudomonas aeruginosa DeltaPA4662 and depositedunder the Budapest treaty before the Spanish Type Culture Collection onApr. 14, 2014 with strain number 8589 by FundaciOn Profesor NovoaSantos.

In a still more preferred embodiment of the third aspect of theinvention, said bacterial strain is the bacterial strain of S. aureusdesignated 132deltamurl/deltadat and deposited under the Budapest treatybefore the Spanish Type Culture Collection on Jun. 11, 2014 with strainnumber 8587 by FundaciOn Profesor Novoa Santos. A fourth aspect of theinvention refers to the bacterial strain as defined in the third aspectof the invention, for use as a medicament, in particular for use as avaccine.

A fifth aspect of the invention refers to the pharmaceutical compositionof the second aspect of the invention or the mutant live auxotrophicbacterial strain for D-glutamate of the third aspect of the invention,for use in a method of prophylactic treatment (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

A sixth aspect of the invention refers to an antibody or fragmentthereof selected from the group consisting of Fab, F(ab′)2, Fv, scFv,di-scFv and sdAB, capable of recognizing a mutant live auxotrophicbacterial strain for D-glutamate, wherein said antibody or fragmentthereof is suitable for the prophylactic treatment (before infection)and/or therapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

An seventh aspect of the invention refers to an antibody or fragmentthereof selected from the group consisting of Fab, F(ab′)2, Fv, scFv,di-scFv and sdAB, obtained or obtainable after immunization of a mammalwith a mutant live auxotrophic bacterial strain for D-glutamate, whereinsaid antibody or fragment thereof is suitable for the prophylactictreatment (before infection) and/or therapeutic treatment (afterinfection or after the clinical manifestation of the disease caused bythe infection) of animals and/or humans against infection with the wildtype form of the mutant auxotrophic bacteria of the composition

In a preferred embodiment of the seventh aspect of the invention, themammal used for the immunization is selected from the group consistingof placental (including humans), marsupial and monotremes.

An eighth aspect of the invention refers to a pharmaceuticalcomposition, preferably a vaccine, comprising the antibodies orfragments thereof of any of the sixth or seventh aspects of theinvention and a pharmaceutically acceptable carrier or diluent andoptionally an adjuvant, wherein said pharmaceutical composition issuitable for the prophylactic (before infection) and/or therapeutictreatment (after infection or after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacteria ofthe composition. In a preferred embodiment of the ninth aspect of theinvention, said pharmaceutical composition is a vaccine wherein saidvaccine optionally comprises an adjuvant.

A ninth aspect of the invention refers to the antibodies or fragmentsthereof of the sixth or seventh aspects of the invention, for use intherapy, in particular for use in passive immunization.

A tenth aspect of the invention refers to the pharmaceutical compositionof the ninth aspect of the invention or the antibodies or fragmentsthereof of any of the sixth or seventh aspects of the invention, for usein a method of prophylactic treatment (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacteria of the composition.

An eleventh aspect of the invention refers to the pharmaceuticalcomposition of the second or eighth aspects of the invention or themutant live auxotrophic bacterial strain for D-glutamate of the thirdaspect of the invention or the antibodies or fragments thereof of any ofthe sixth or seventh aspects of the invention, for use in a method ofprophylactic treatment (before infection) and/or therapeutic treatment(after infection or after after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacteria ofthe composition and wherein said composition, bacterial strain orantibody or fragment thereof is administered intranasally,intradermally, subcutaneously, orally, by aerosol, intramuscularly, wingweb and eye-drop administration.

In this sense, variation in IgG sera levels were observed for the threeD-glutamate auxotrophic strains of A. baumanii, P. aeuginosa and S.aureus species, these levels being dependent on whether the vaccine isadministered by intraperitoneal, intramuscular, subcutanous orintranasar route. Likewise, the vaccine schedule of administration(doses composition and frequency of administration) can affect the finallevels of antibodies produced (see example 34). Therefore, determiningthe most appropriate schedule and route of vaccination for optimalantibody titers may need to be determined for each pathogen. For thispurpose, to correlate the humoral response obtained by administrationroutes other than intraperitoneal (not routinelly used) with protectiveefficacy, the authors evaluated the use of ΔPA4662 vaccine strainadministrated via intramuscular (preferred route for administration inhumans) because this one elicited similar high level of IgGs as theintraperitoneal route (see example 18). In this regard, mice werechallenged with P. aeruginosa PAO1 wild type as before. After thechallenge, the authors observed 100% mortality in the group ofnon-vaccinated mice whereas all vaccinated mice showed 100% survival(see FIG. 50). This result suggests that vaccination using theintramuscular route of administration is at least as effective as theintraperitoneal route.

In addition, it is noted that the medicament, pharmaceutical compositionor vaccine composition of the present invention can be used both inasymptomatic patients as well as in those who have already shownsymptoms of the disease.

Furthermore, the authors of the present invention have surprisinglyfound that by using a kit or device comprising an antibody or fragmentthereof of the invention, the kit or device permits a reliablequalitative and/or quantitative analysis of bacterial species in abiological of sample of a subject and, in particular, in the plasma ofsubjects suspected of suffering from a disease of bacterial origin.

Therefore a further aspect of the invention, namely a twelfth aspect ofthe invention, relates to a kit or device comprising an antibody orfragment thereof of the invention for use in the qualitative and/orquantitative determination of bacterial species in a biological samplefrom a mammal, in particular, in the plasma of a mammal suspected ofsuffering from a bacterial disease.

A preferred embodiment of the twelfth aspect of the invention relates toa kit for detecting an infection of bacterial origin through animmunoassay comprising:

-   -   (i) a first antibody called “capture antibody” obtain or        obtainable according to the sixth or seventh aspect of the        invention capable of recognizing the bacterial species causing        the infection, wherein said first antibody is preferably        attached to a solid support;    -   (ii) a second labeled antibody called “detection antibody” which        recognizes a region other than the region recognized by the        first antibody, wherein said second antibody comprises a marker        which may be fluorescent, luminescent or an enzyme;    -   (iii) a reagent showing affinity for the second antibody, said        reagent being coupled to a first member of a binding pair; and    -   (iv) a second member of a binding pair coupled to a fluorescent,        luminescent or an enzyme, wherein the binding pair is selected        from the group consisting of: hapten and antibody; antigen and        antibody; biotin and avidin; biotin and streptavidin; a biotin        analogue and avidin; a biotin analogue and streptavidin; sugar        and lectin; an enzyme and a cofactor; a nucleic acid or a        nucleic acid analogue and the complementary nucleic acid or        nucleic acid analogue.

In the context of the present invention, the first antibody is called“capture antibody”, which means that this antibody is used to retrievefrom a sample all bacterial species to which the antibody specificallybinds. There is practically no limitation on the type of antibody thatcan be used as a capture antibody provided that it has been obtainedaccording to the seventh aspect of the invention. Antibodies suitablefor use as capture antibodies include but are not limited to thefollowing: “intact antibodies”, “Fab” fragments, “F(ab′)2 fragments,“Fv” fragments, single chain Fv fragments or “scFv”, “Diabodies” and“bispecific antibodies” (Bab)

All these antibody fragments can be further modified using conventionaltechniques known in the art, for example, by using deletion(s),insertion(s), substitution(s), or addition(s) of amino acid and/orrecombination(s) and/or any other modification(s) (e.g.posttranslational and chemical modifications, such as glycosylation andphosphorylation changes) known in the art either.

Antibodies suitable as capture antibodies include both polyclonal andmonoclonal antibodies. For production of polyclonal antibodies varioushosts can be immunized including goats, rabbits, rats, mice, camels,dromedaries, llamas, humans, birds and others. Depending on the hostspecies, various adjuvants may be used to increase the immunologicalresponse.

For production of monoclonal antibodies, conventional techniques can beused. For example, monoclonal antibodies can be obtained using thehybridoma method first described by Kohler et al, Nature, 256:495 (1975)using the procedure described in detail in units 11.4 to 11.11 ofAusubel, F M et al. (Current Protocols in Molecular Biology, John Wiley& Sons Inc.; rings edition, 2003). Alternatively, monoclonal antibodiesmay be isolated by recombinant DNA from phage antibody librariesgenerated using the techniques described in McCafferty et al, Nature348:552-554 (1990). Clacksoii at al, Nature, 352: 624-628 (1991) andMarks at a!, J. Mol. Biol, 222:581-597 (1991) describe the isolation ofmurine and human antibodies, respectively, using phage libraries.Subsequent publications describe the production of high affinity humanantibodies (in the nM range) by chain shuffling (Marks et al, BiolTechnology, 10:779-783 (1992)), as well as combinatorial infection andin vivo recombination as a strategy for constructing very large phagelibraries (Waterhouse at al, Nucl Acids Res, 21: 2265-2266 (1993)).Thus, these techniques are viable alternatives to traditional hybridomatechniques of monoclonal antibodies for the isolation of monoclonalantibodies.

A thirteenth aspect of the invention refers to the use of the kit ordevice of the twelfth aspect of the invention, for the qualitativeand/or quantitative determination of bacterial species or bacterialstrains in a biological sample from a mammal, in particular, in theplasma of a mammal suspected of suffering from a bacterial disease.

Lastly, a fourteenth aspect of the present invention refers to a methodof cultivation of bacterial strains auxotrophic for D-glutamatecomprising the utilization of different concentrations of D-glutamate.

In a preferred embodiment of the fourteenth aspect of the invention, therange of concentration of D-glutamate is 0.00001-120 mM.

In a still more preferred embodiment of this aspect of the invention,said range of concentration of D-glutamate is 0.01-50 mM.

Even more preferably, said range of concentration of D-glutamate is10-20 mM.

Lastly, a further aspect of the invention refers to killed mutantauxotrophic bacterial strain for D-glutamate or to immunologicalfragments of the killed bacteria thereof as well as to the use of thesebacteria or to its inmunological fragments thereof for the usesdescribed in the present invention.

The purpose of the following examples is merely to illustrate theinvention and not to limit the same.

EXAMPLES Example 1 Analysis and Identification of the Nucleotides and ofthe Amino Acid Sequences of the Genes Encoding the A. baumannii ATCC17978 Glutamate Racemase Enzyme

The authors of the present invention conducted a search for genesencoding glutamate racemase enzyme in the A. baumannii ATCC 17978 genomeannotation using the Protein Knowledgebase (UniProtKB) database searchtool. Two amino acid sequences corresponding to proteins Murl (locusA1S_0380 and locus A1S_3398) were identified. These sequences werecompared to one another, and to other glutamate racemase proteinsequences present in other bacterial species the genomes of which weresequenced, by means of the Clustal Omega alignment tool.

As a result of the previous analysis the genes encoding glutamateracemase protein in the A. baumannii ATCC 17978 genome were identified.Two candidate genes for possible glutamate racemases were thus found:the gene annotated as A1S_0380 encoding a 288 amino acid protein calledmurl and gene A1S_3398 encoding a 266 amino acid protein also calledmurl.

FIG. 4 shows the alignment of the amino acid sequences of differentglutamate racemase proteins, including both Murl (locus A1S_0380) andMurl (locus A1S_3398) proteins of A. baumannii ATCC 17978 and theglutamate racemase genes of E. coli, strain K12, and P. aeruginosa,strain PAO1. Murl protein encoded in locus A1S_0380 and Murl proteinencoded in locus A1S_3398 of A. baumannii ATCC 17978 share a 29.8%similarity with one another on the amino acid sequence level, and theyhave a 33.2% and 23.2% similarity with Murl protein of E. coli (strainK12), respectively, and 35.8% and 37.5% similarity with Murl protein ofP. aeruginosa (strain PAO1), respectively.

Example 2 Construction and Characterization of Different A. baumanniiMutant Strains without Glutamate Racemase Genes

Homologous double recombination was carried out using suicide vectorpMo130 to construct the different mutant strains. First, the A1S_0380murl and A1S_3398 murl genes were deleted independently, both mutantstrains called Δ0380 and Δ3398, respectively, being obtained. Fragmentsof about 1.000 bps corresponding to the upstream and downstream regionsof each of the genes were amplified by means of PCR to construct thesemutants. Both fragments were cloned into vector pMo130 using theresulting recombinant plasmid to remove the A1S_0380 murl and A1S_3398murl genes by means of homologous double recombination.

The A1S_0380 murl gene upstream fragment was obtained by means of PCRamplification with oligonucleotides UP0380(NotI)F and UP0380(BamHI)R,the resulting PCR product being digested by NotI and BamHI restrictionenzymes. The A1S_0380 murl gene downstream fragment was obtained bymeans of PCR amplification with oligonucleotides DOWN0380(BamHI)F andDOWN0380(SphI)R, the resulting PCR product being digested with BamHI andSphI restriction enzymes. Digested locus A1S _0380 murl gene upstreamand downstream fragments were ligated into vector pMo130, which ispreviously linearized with NotI and SphI enzymes, recombinant plasmidcalled pMo130UP/DOWN0380 being obtained.

Recombinant plasmid pMo130UP/DOWN3398 was obtained in the same way as inthe preceding case. The oligonucleotides used in this case wereUP3398(Notl)F_II and UP3398(BamHI)R_II for upstream fragmentamplification and oligonucleotides DOWN3398(BamHI)F and DOWN3398(SphI)Rfor downstream fragment amplification of locus A1S_3398 murl gene.

Plasmids pMo130UP/DOWNO380 and pMo130UP/DOWN3398 were introduced in E.coli TG1 by electroporation, The different TG1 strains transformed witheach of the preceding plasmids were cultured in LB with kanamycin (50μg/mL) for 18 h at 37° C. The obtained colonies were sprayed withpyrocatechol (0.45 M) and only the yellow colonies, expressing xylEreporter gene of plasmid pMo130, were selected. OligonucleotidesUP0380(NotI)F and DOWN0380(SphI)R were used for simultaneous upstreamand downstream fragment amplification of plasmid pMo130UP/DOWN0380introduced by electroporation in yellow kanamycin-resistant TG1colonies. In turn, oligonucleotides UP3398(NotI)F_II and DOWN3398(SphI)Rwere used for simultaneous upstream and downstream fragmentamplification of plasmid pMo130UP/DOWN3398.

Plasmids pMo130UP/DOWN0380 and pMo130UP/DOWN3398 obtained from thetransformed TG1 strains were individually introduced in A. baumanniiATCC 17978 by electroporation, as described above. The co-integrantcolonies were selected in LB medium agar with kanamycin (50 μg/mL) andsprayed with pyrocatechol (0.45 M) for selecting the yellow colonies(XyIE⁺). For the subsequent individual resolution of the co-integrantcolonies, these were inoculated in 1 mL of LB medium and grown for atleast 4 hours at 37° C. under stirring. The cultures were seriallydiluted and seeded in LB agar containing 15% sucrose (the sacB genecontained in plasmid pMo130 confers sensitivity to sucrose). Theresulting colonies were sprayed with pyrocatechol, and white colonies(resolved co-integrants) were analyzed by PCR to confirm Δ0380 and Δ3398deletions, produced by the allelic exchange of plasmidspMo130UP/DOWN0380 and pMo130UP/DOWN3398 with alleles A1S_0380 andA1S_3398, respectively. Oligonucleotides EXTFW0380 with EXTRV0380 andINTFW0380 with INTRV0380 were used to confirm the Δ0380 mutation.Oligonucleotides EXTFW3398 with EXTRV3398 and INTFW3398 with INTRV3398were used to confirm the Δ3398 mutation.

The double mutant (Δ0380/Δ3398) was constructed following the previouslydescribed protocol for constructing single mutants. PlasmidpMo130UP/DOWN3398 was introduced in mutant Δ0380 by electroporation, andthe co-integrant colonies were resolved as was previously done, exceptthe colonies were grown in LB agar with and without 10 mM D-glutamate toidentify the possible A. baumannii ATCC 17978 double mutants Δ0380/Δ3398since the colonies with the double mutation require this compound togrow.

The presence of locus Δ0380 in mutant Δ0380, locus Δ3398 in mutant Δ3398and both loci Δ0380 and Δ3398 in the double mutant, as well as theabsence of the corresponding wild type loci, was confirmed by means ofPCR with the following oligonucleotide pairs: EXTFW0380/EXTRV0380,INTFW0380/INTRV0380, EXTFW3398/EXTRV3398, INTFW3398/INTRV3398,UP0380(NotI)F/DOWN0380(SphI)R, UP3398(NotI)F_II/DOWN3398(SphI)R, asappropriate in each case.

FIG. 5 shows the results of the PCR performed to confirm the differentmutations present in the three constructed A. baumannii ATCC 17978mutant strains. The presence of the wild type loci A1S_0380 murl orA1S_3398 murl or the respective mutant variants, Δ0380 and Δ3398, ineach of the new mutant strains constructed (Δ0380, Δ3398 andΔ0380/Δ3398), was confirmed by PCR with the oligonucleotides mentionedabove.

The culture of the different mutant strains in the presence and absenceof D-glutamate showed that the loss of a single gene encoding glutamateracemase protein in A. baumannii ATCC 17978 does not affect growth ofthis bacterium. However, double mutant Δ0380/Δ3398 is auxotrophic,requiring the exogenous addition of D-glutamate in the culture medium toallow growth. FIG. 6 shows the method of selecting colonies with thedouble mutant Δ0380/Δ3398 genotype. This strain only grows in LB agarsupplemented with D-glutamate. This characteristic is used to selectco-integrant colonies with individual Δ0380 mutation, which does notrequire D-glutamate to grow.

In summary, the obtained results demonstrate that the presence of any ofthe two wild type loci studied, i.e., both A1S_0380 murl and A1S_3398murl, is sufficient for normal growth in LB agar without added0-glutamate, and that the simultaneous deletion of both genes make thisstrain unable to grow in LB agar. It is in turn demonstrated that theA1S_0380 murl and A1S_3398 mud genes of A. baumannii ATCC 17978 are theonly genes responsible for the production of D-glutamate in this strain.

Example 3 Effect of D-glutamate on Double Mutant (Δ0380/Δ3398) Growthand Viability in Liquid Culture Medium

To determine the growth and viability curve, A. baumannii wild typestrain ATCC 17978 and the double mutant strain were cultured for 18 h at37° C. in LB supplemented with 10 mM D-glutamate. The bacterial cultureswere centrifuged and the pellets washed twice with LB and adjusted toOD_(600 nm)=0.1. 100 μL were subsequently inoculated in 100 mL liquid LBwith and without 10 mM D-glutamate, and these cultures were incubated at37° C. under stirring, taking samples every 60 minutes for 7 hours, andfinally, after 24 hours, to determine optical density of the medium. Inparallel, samples were taken every 2 hours up to 6 hours, and finally,another sample was taken after 24 hours to determine the CFU (ColonyForming Units) in LB agar with 10 mM D-glutamate. All cultures were madein triplicate.

Growth curves for wild type strain ATCC 17978 as well as for mutantstrain Δ0380/Δ3398 were made to evaluate the effect of the absence ofD-glutamate as a function of time and the viability of these strains inthe presence and absence of this compound. Complete absence of bacterialgrowth of double mutant Δ0380/Δ3398 in culture medium withoutD-glutamate (FIG. 7A) was observed. However, the growth of this strainin the presence of D-glutamate was similar to the growth of thewild-type strain in the same conditions. When analyzing cell viabilityfor 6 hours of double mutant Δ0380/Δ3398 in the absence of D-glutamateit was observed that unlike the wild-type strain, double mutantviability drops significantly (1 log10) in the first 4 hours of culturedue to the limitation of this compound (FIG. 7B).

Example 4 Morphological Analysis of A. baumannii Wild Type Strain ATCC17978 and Double Mutant Strain (Δ0380/Δ3398) by Means of ElectronMicroscopy

To take microphotographs by scanning electron microscopy (SEM), the A.baumannii wild type strain ATCC 17978 and double mutant strain werecultured for 18 h at 37° C. in LB supplemented with 10 mM D-glutamate.The bacterial cultures were centrifuged, the pellets washed twice with0.1% NaCl and suspended in LB. 50 μL of the last cultures wereinoculated in 5 mL of LB with D-glutamate at concentrations of 0; 0.1;1.25 and 10 mM. The cultures were incubated at 37° C. under stirring for2 hours and were subsequently centrifuged and washed twice with PBS. Thepellets were then fixed with 4% paraformaldehyde in 0.1 M PBS pH 7.4 for30 min. After fixing, the samples were washed again twice with PBS, andeach sample was dehydrated in increasing series of ethanol (50%, 70%,90% and 100%) for 10 minutes each. The samples were then dried to thecritical point with CO₂ (Bal-Tec CPD 030), fixed in aluminum supports,and coated with a layer of gold (Bal-Tec SCD 004 sputter coater).Observation was conducted and photographs were taken using a JeolJSM-6400 transmission electron microscope.

To take microphotographs by transmission electron microscopy (TEM),double mutant Δ0380/Δ3398 was cultured in LB agar supplemented with 10mM D-glutamate for 18 h at 37° C. Subsequent passage to MH agar wasperformed and it was incubated at 37° C. for 18 h. After incubation, 2-3colonies were dissolved in PBS buffer, the suspension was centrifugedand the resulting pellet was washed first with cacodylate buffer, andimmediately after that the cells were fixed in cold 2.5% glutaraldehydeprepared in 0.2 M sodium cacodylate buffer pH 7.4 for 4 hours at roomtemperature. The pellets were then washed with cacodylate buffer,dehydrated in acetone and embedded in SPURR (Spurr's Epoxy EmbeddingMedium). Ultrathin sections (70 nm) of these samples were obtained andthey were stained with uranyl acetate and lead citrate for observationunder a JEOL JEM 1010 (80 kV) electron transmission microscope.

At the microscopic level, significant morphological and structuralchanges were observed in strain Δ0380/Δ3398 as the exogenous supply ofD-glutamate decreases. FIG. 8 shows the microphotographs taken with ascanning microscope of both strains cultured in the presence ofdifferent concentrations of D-glutamate. Therefore, in FIG. 8A, it canbe seen how double mutant Δ0380/Δ3398 is unable to divide in the absenceof D-glutamate. The bacterial cells seen in the figure with 0 mMD-glutamate are reminiscent of the inoculum previously grown with thiscompound. In the presence of 0.1 mM D-glutamate, the bacterial cellsshow some growth, though it is very slow and very peculiar with anirregular division pattern, in which they form filamentous aggregates ofcells with atypical binary fission. A mass of protoplasts is alsoobserved (the absence of cell wall leaves the bacteria protoplasmsurrounded by only the cell membrane, the protoplast). In the presenceof 1.25 mM D-glutamate greater cell density is seen with respect to thepreceding condition (reflection of a higher growth rate) but with manyprotoplasts. Some cell aggregates with atypical binary fission continueto be visible, and, though lower in number, part of the cells now has anappearance similar to their wild-type morphology. Finally, in mediumsupplemented with 10 mM D-glutamate, all the cells have an appearancesimilar to their wild-type homologue, both at the density level and atthe cell morphology level. No atypical division is observed.

Furthermore, as can be seen in FIG. 8B, the authors of the presentinvention took several microphotographs in the bacterial preparationswith 0.1 mM D-glutamate, on different scales, both of double mutantΔ0380/Δ3398 and of the wild-type strain. This latter strain has typicalGram-negative coccobacillus morphology and a regular cell divisionpattern, with typical binary fission, a high cell density beingobserved. In the case of the double mutant, the preceding morphologiesand atypical cell divisions are observed, with a subsequent low celldensity. The presence of protoplasts is evident, as they are alwaysvisible in the vicinity of the structurally intact cell forms, arisingas derivations or “ghosts” of the previous ones. Within these complexes,the appearance of the cell wall is different in comparison with thewild-type strain because the cell surface is rough and irregular. Theprofound alteration at the division level is also evident because alarge amount of cell filaments consisting of 3 or more units isobserved.

As with the electron scanning microscopy, transmission electronmicroscopy studies were performed and they show that the cell wall ofdouble mutant Δ0380/Δ3398, when kept in the absence of D-glutamate,experiences progressive destruction as a result of the inactivation ofthe glutamate racemase protein function, cell lysis and subsequentbacterial death taking place. FIG. 9 shows different stages of cell walldegeneration, ranging from cells with an altered conformation that losetheir semi-rigid structure to cells showing several ruptures anddisplacement of the outer membrane, lysis and extrusion of theintracellular content (especially genetic material). Cells stillmaintaining the inner membrane (remnants of the inoculum grown withD-glutamate) and having the genetic material dispersed therein are alsoseen because glutamate racemase is an enzyme that can act in themodulation of DNA gyrase. Finally, the mechanism of bacterialdestruction is as follows: the absence of cell wall leaves the bacteriaprotoplasm surrounded by only the cell membrane (protoplast), whichmakes this strain an organism that is extraordinarily sensitive tovariations in tonicity of the medium. Then the phenomenon of clearingoccurs, which takes place when the protoplasts explode and leavecytoplasmic membrane residues called “ghosts”, membrane and liposomeaggregates also being generated.

Example 5 Determining the Lethal Dose (LD) of A. baumannii Wild TypeATCC 17978 and Double Mutant Strain Δ0380/Δ3398 in BALB/c Mice in anAcute Infection Model

The authors of the present invention produced systemic infection inBALB/c mice through the intraperitoneal administration of an inoculum insaline of A. baumannii wild type strain ATCC 17978 and double mutantstrain Δ0380/Δ3398. To prepare the inocula, the bacteria were culturedin LB (wild type strain) and LB +10 mM D-glutamate (double mutant) at37° C. under shaking until reaching OD_(600 nm)=0.7 (this inoculum iscalled 1×). The cultures were centrifuged, and the bacterial pelletswere washed twice with LB. Once washed, the bacterial suspensions wereadjusted according to the previous OD_(600 nm) to different doses (0.1×;1×; 2×; 2.5×; 3×; 4×; 6×; 8× and 10×) with 0.9% NaCl and inoculated (100μL) in female BALB/c mice intraperitoneally (1× being understood as thebacterial inoculum with OD_(600 nm)=0.7; 0.1× being understood as thebacterial inoculum 1× diluted 10-fold; 2× being understood as thebacterial inoculum 1× concentrated two-fold; and so on and so forth).The mice were monitored for 7 days post-infection to determine survivalwith the different doses. The lethal doses (LD) were determinedaccording to survival observed in both cases, LD₁₀₀ being understood asthe minimum lethal dose for 100% of the mice.

FIG. 10A shows different degrees of survival in mice when they areinfected with increasing doses of the wild type strain. A gradualdecrease in survival of the mice is observed with respect to the 2×dose, the LD₁₀₀ (2.5×) being able to be determined, and LD₁₀₀ beingunderstood as the minimum dose necessary to reduce survival of mice to0%. When the administered dose is greater than 3×, a more rapidreduction in survival is observed.

FIG. 10B shows different degrees of survival in mice infected withincreasing doses of double mutant Δ0380/Δ3398. In the case of thisstrain, LD₁₀₀ is 6×, a very high dose of bacterial inoculum, indicatingthat this strain is less virulent. Like in the case of the wild typestrain, when the bacterial dose administered is greater than LD₁₀₀, amore rapid reduction in survival of the mice is observed.

Example 6 Determining the Bacterial Load in the Liver in a SystemicInfection Model with the A. baumannii Wild Type Strain ATCC 17978 andMutant Strains Δ0380, Δ3398 and Δ0380/Δ3398 in BALB/c Mice

As described above, a systemic infection was established in BALB/c micethrough the intraperitoneal administration of an inoculum in saline ofA. baumannii wild type strain ATCC 17978 and mutant strains Δ0380, Δ3398and Δ0380/Δ3398. The wild type strain and single mutants Δ0380 and Δ3398were cultured in LB, and double mutant Δ0380/Δ3398 in LB +10 mMD-glutamate at 37° C. under shaking until reaching OD_(600 nm)=0.7 (1×).The cultures were centrifuged, and the bacterial pellets were washed zotwice with LB. Once washed, the bacterial suspensions were adjustedaccording to the previous OD_(600 nm) to the 2× dose with 0.9% NaCl andinoculated (100 μL) in male BALB/c mice (n=8-9) intraperitoneally. Themice were sacrificed with sodium thiopental at 12 hours post-infection.Livers were extracted and weighed aseptically, and after beinghomogenized in 0.9% NaCl, the CFU per gram of liver were determined inLB agar (wild type strain, Δ0380 and Δ3398) and LB agar with 10 mMD-glutamate (double mutant Δ0380/Δ3398).

It is observed in this acute infection model that the infection takesplace with a rapid spread through the blood, causing the mice to diebetween 11 and 30 hours post-infection (FIG. 10A). Based on the bacteriacount in the organs, the authors of the present invention can obtain ameasurement of the invasive and replicative capacity of the differentstrains. The following average values were thus obtained: 8.29 Log₁₀CFU/g (mice infected with the wild type strain); 6.88 Log₁₀ CFU/g (miceinfected with strain Δ0380); 8.06 Log₁₀ CFU/g (mice infected with strainΔ3398) and 1.59 Log₁₀ CFU/g (mice infected with strain Δ0380/Δ3398)(FIG. 11).

Based on these values, significant differences were observed (P<0.0001,Mann-Whitney U test) in the counts of the strains Δ0380 and Δ0380/Δ3398compared to the wild type strain. The most drastic and evident reductionwas observed when analyzing the colony count of double mutant strainΔ0380/Δ3398, in which a reduction of almost 7 log values were obtainedin the average bacterial load and 44.4% of animals from the organs ofwhich bacteria was not recovered.

Example 7 Determining the Bacterial Load in the Liver, Spleen, and Lungin a Systemic Infection Model with A. baumannii Wild Type Strain ATCC17978 in BALB/c Mice Pre-Immunized with Double Mutant Δ0380/Δ3398

To evaluate the efficacy (level of protection) of strain Δ0380/Δ3398 asa vaccine, male BALB/c mice (n=10) were immunized intraperitoneally (100μL) with strain Δ0380/Δ3398 in saline at 1× dose on days 0 and 14. Agroup of control mice (n=10) was administered 100 uL of saline in anidentical manner on days 0 and 14. On day 21, the mice were infectedwith a 4× inoculum of A. baumannii wild type strain ATCC 17978 byintraperitoneal injection. The mice were sacrificed with sodiumthiopental 12 hours post-infection. The liver, spleen and lung of eachof the mice were processed aseptically, and the CFU were determined, asdescribed above. The bacterial inocula were prepared and adjusted asdescribed above.

The protective effect of the vaccination with the double mutantΔ0380/Δ3398 was thus confirmed when it was observed thatpre-immunization with this strain causes a very significant reduction inthe bacterial load of different organs of mice infected with a lethaldose of A. baumannii ATCC 17978. In fact, a severe reduction in thebacterial load of each of the organs of immunized mice was observed incomparison with the bacterial load of non-immunized mice (P<0.0001,Mann-Whitney U test) (FIG. 12).

Example 8 Quantification of IgG Antibodies Against A. baumannii ATCC17978 Through Indirect ELISA in BALB/c Mice Subjected to DifferentVaccination Regimens with Strain Δ0380/Δ3398

To evaluate the antibody-mediated immune response at differentvaccination regimens, male BALB/c mice (n=12) were immunized throughintraperitoneal injection (100 μL) of strain Δ0380/Δ3398 in saline at 1×dose on days 0 and 14. A group of control mice (n=12) was administered100 μL of saline in an identical manner on days 0 and 14. On day 7post-immunization, sera were obtained from 12 of the mice immunized witha single dose of the vaccine (administered on day 0), and on day 21,sera were obtained from the remaining mice immunized with two doses ofthe vaccine (administered on days 0 and 14) (n=12) together with thesera of the control mice (n=12), injected with saline. To obtain thesera, the mice were anesthetized with sodium thiopental and blood wasdrawn through puncture of the retro-orbital plexus. The sera wereseparated from the blood cells by centrifugation and stored at −80° C.until subsequent analysis thereof. 1×, 0.5×, 0.1×, 0.05× and 0.01× dosesof vaccine were prepared in an identical manner, and the mice (n=6/dose)were immunized on days 0 and 14, as was previously done. On day 21, serawere obtained from the immunized mice and from the control mice (0×),injected with saline.

IgG quantification was performed by means of an indirect Enzyme-linkedImmunosorbent Assay (ELISA). 96-well ELISA plates were “coated” with A.baumannii wild type strain ATCC 17978 which was fixed to the bottom ofthe wells after 18 h of incubation at 4° C. in 100 mMcarbonate-bicarbonate buffer, pH 9.6. Five (5) washings were performedwith phosphate buffered saline (PBS) to remove the unfixed bacteria. Theresidual sites were blocked by means of incubating at 25° C. for 1 hwith 200 μL per well of blocking solution (5% skim milk in PBS). Thecontent of the plates was aspirated and washed 5 times with washingbuffer (0.005% Tween 20 in PBS). The plates were incubated for 1 h at25° C. with 100 φL of the sera serially diluted in dilution buffer (DMEMculture medium with 5-10% FCS). Five (5) washings were performed withwashing buffer to remove the antibodies that have not reacted. 100 μLper well of secondary antibody (peroxidase-labeled mouse IgG) diluted1/5000 in dilution buffer were added. It was incubated for 1 h at 25° C.in the dark. The plates were washed 5 times with washing buffer toremove the labeled anti-antibodies that did not react. Development wasperformed by means of incubation for 3 min with 100 μL of TMB substrate(peroxidase substrate). The reaction was stopped with 50 μL of 1 M HClper well. The colorimetric reading was taken at 450 nm. To determine theIgG titers in each case, the endpoint titer, the maximum serum dilutionhaving a value that exceeds the blank absorbance reading (absorbance ofthe dilution buffer) by 0.1 values, is sought.

As discussed above, the blood samples collected from each mouse wereused to determine the antibody (IgG) titer by means of the ELISAtechnique with respect to A. baumannii ATCC 17978 and thus measure thecapacity of the vaccine to generate an immune response. Significantlevels of antibodies against the wild type strain were detected in allthe immunized mice with respect to non-vaccinated mice (FIG. 13).Antibody production was significantly higher both on day 7 and on day 21(after one injection and after two injections of strain Δ0380/Δ3398,respectively) compared to non-vaccinated mice. Furthermore, antibodyproduction on day 21 (after 2 successive injections in time with strainΔ0380/Δ3398) was significantly greater than antibody production on day 7(a single injection of strain Δ0380/Δ3398).

On the other hand, the efficacy of different doses of strain Δ0380/Δ3398was determined in order to determine the minimum dose necessary tostimulate the mouse immune system. To that end, groups of 6 mice wereimmunized on days 0 and 14 with is each of the following doses: 0.5×,0.1×, 0.05× and 0.01× (2, 10, 20 and 100-fold lower, respectively).Significant differences between IgG antibody production on day 21 wereobserved between the group of mice immunized with the 0.01× dosecompared to mice in the control group (P<0.01, Mann-Whitney U test)(FIG. 14), although to a lesser extent than the higher vaccine dose.This demonstrates that a dose 100-fold lower than the 1× dose is enoughto trigger IgG production in mice, demonstrating the efficacy of thisstrain as a vaccine.

Example 9 Cross-Reactivity Assay with IqG Antibodies of BALB/c MiceImmunized with Strain Δ0380/Δ3398 with Respect to A. baumannii ATCC17978, A. baumannii ATCC 19606 and A. baumannii AbH12O-A2

ELISA was performed with respect to A. baumannii ATCC 19606 and A.baumannii AbH12O-A2 with the previously obtained sera to evaluate theantibody-mediated immune response in BALB/c mice immunized with strainΔ0380/Δ3398 and thus measure the capacity of the vaccine to generate abroad immune response. It must be pointed out that Acinetobacterbaumannii strain AbH12O-A2 is a highly invasive strain which wasisolated in a hospital outbreak that caused several patients to die, andis characterized by its resistance pattern with respect to multipleantibiotics. To that end, plates with A. baumannii strain ATCC 17978, A.baumannii strain ATCC 19606 and A. baumannii strain AbH12O-A2 were“coated” as described above. Five (5) washings were performed with PBSand subsequent blocking was performed with 5% skim milk in PBS. Thecontent of the plates was aspirated and washed 5 times with washingbuffer. The plates were incubated for 1 hour at 25° C. with 100 μL ofthe test sera diluted ⅕ in dilution buffer. Five (5) washings wereperformed with washing buffer and 100 μL per well of peroxidase-labeledmouse IgG diluted 1/5000 were added. It was incubated for 1 h at 25° C.in the dark. The plates were washed 5 times and development wasperformed by means of incubating for 3 minutes with 100 μL of TMBsubstrate. The reaction was stopped with 50 μL of 1 M HCl per well andthe color developed at 450 nm was read. The endpoint titer wasdetermined in each case as described above.

Results similar to those observed with respect to A. baumannii strainATCC 17978 were obtained with respect to A. baumannii strain ATCC 19606and A. baumannii strain AbH12O-A2 (FIG. 15), similar antibody titersbeing observed and demonstrating that immunization with strainΔ0380/Δ3398 not only generates antibodies against the isogenic wild typestrain, but also generates IgG antibodies against other strains withdifferent resistance and virulence patterns such as strain ATCC 19606and strain AbH12O-A2.

Example 10 Protection of BALB/c Mice Against Challenge with Different A.baumannii Strains by Immunization with the Δ0380/Δ3398 Mutant

To evaluate the efficacy of the mutant strain Δ0380/Δ3398 as a vaccine,BALB/c mice (n=6-12) were administered 100 μL of Δ0380/Δ3398 strain (1×*dose in saline) on days 0 and 14. Control mice were administered onlysaline identically at days 0 and 14. Twenty one days after the secondinjection, mice were challenged with A. baumannii strains ATCC 17978 (4×dose in saline), AbH12O-A2 (4× dose in saline) and Ab307-0294 (0.75×),independently, in order to establish a lethal systemic infection in bothcases (100 μL of intraperitoneal injection). After the challenge, micewere monitored for 7 days to determine the survival rate of vaccinatedmice compared to control mice (unvaccinated).

*in the case of the challenge with Ab307-0294 strain, vaccinated micewere administered 0.1× and 1× doses of the Δ0380/Δ3398 strain,respectively, on days 0 and 14.

When infected with a 4× dose of the A. baumannii ATCC 17978 strain, 11deaths were observed in the group of unvaccinated mice during the first24 hours, which means a mortality rate of 92% in this group (n=12). Incontrast, all vaccinated mice (n=12) survived to the challenge,overcoming the infection, which means a 100% survival (see FIG. 16) ratein this group. Differences in survival between the two groups werehighly statistically significant (P<0.0001, according to the Mantel-Coxlog-rank test).

Furthermore, it was determined whether the response produced byimmunization with the Δ0380/Δ3398 strain was sufficient to provideprotection from lethal infection with other A. baumannii strains,including highly virulent and pathogenic strains. In the case ofchallenge with the AbH12O-A2 strain, 9 deaths were observed in the groupof unvaccinated mice during the first 19 hours, which means a mortalityrate of 100% (n=9). On the other hand, all vaccinated mice survived(n=9; 100% survival rate) (see FIG. 17). Differences in survival betweenthe two groups were highly statistically significant (P<0.0001,according to the Mantel-Cox log-rank test).

In the case of challenge with the Ab307-0294 capsulated strain, werecorded a 100% mortality rate in the group of unvaccinated mice withinthe first 24 hours and a 83% survival rate in the group of micepreviously immunized with the Δ0380/Δ3398 strain (see FIG. 18). Thisconfirms that vaccination with the mutant strain confers protectionagainst a systemic infection caused by an A. baumannii strain withmarked virulence. Differences in survival between the two groups werehighly significant (P<0.0022, according to the Mantel-Cox log-ranktest).

All these results suggest that vaccination with the Δ0380/Δ3398 straincan provide protective immunity against infection with a diverse groupof A. baumannii strains.

Example 11 Environmental Safety Assessment of the Δ0380/Δ3398Strain—Evaluation of Water Osmolisis

The live attenuated bacterial strain constituting the active ingredientof a vaccine candidate should be incapable of replicating and persistingin the general environment once it leaves the vaccinated individual. Tocompare the ability of A. baumannii ATCC 17978 wild type and the mutantstrain Δ0380/Δ3398 to be long-time traced in the general environment, weevaluated survival of these strains in water without any contribution ofnutrients or salts, at 37° C. and under agitation (180 rpm) conditionsfor the time necessary to observe the loss of viability by cellularosmolisis. Daily samples of the culture were taken for 2 days andfinally two times per week, for the determination of CFU counts in LBagar (wild type strain) and LB agar supplemented with 10 mM D-glutamate(mutant strain). All cultures were performed in triplicate.

A significant decrease in the viability of the mutant strain Δ0380/Δ3398was observed, as no viable bacteria were recovered within and after 5days of culture. in contrast, its wild counterpart, the wild typestrain, remained viable until the 29^(th) day of culture, being totallyirrecoverable at day 40 (see FIG. 19). Differences in survival betweenthe two strain were highly significant (P=0.0061, according to Student'st test).

Example 12 Evaluation of the Stability of the Auxotrophic Phenotype inthe Δ0380/Δ3398 Strain

To test the irreversibility of the nutritional auxotrophy of A.baumannii Δ0380/Δ3398 for the compound D-glutamate, Δ0380/Δ3398 strainwas grown in 100 mL of LB supplemented with 10 mM D-glutamate in optimalconditions for 8 days at 37° C. under agitation conditions (180 rpm).Samples from this culture were taken at the beginning of the incubationperiod and at days 1, 2, 7 and 8 for the determination of CFU in LB agarand LB agar supplemented with 10 mM D-glutamate. All cultures wereperformed in triplicate. In the hypothetical case of a phenotypereversion, similar bacterial counts should be recovered in agar platesover time, independently of the presence or absence of the compound inthe medium. In contrast, we observed significant differences between thebacterial counts obtained when the culture was plated on agar mediumwith and without D-glutamate.

Resulting bacterial counts were significantly higher in the first case(agar plates supplemented with D-glutamate), at the initial stage ofincubation (0 days) and on days 1, 2, 7 and 8 (see FIG. 20) (P=0.0006,according to Student's t test). The recovery of a significantly lowernumber of colonies in the agar plates without D-glutamate can be due toa residual growth derived from the accumulation of this compound in thecytoplasm of bacterial cells during growth in supplemented media. Thisdifference indicates that Δ0380/Δ3398 strain remains auxotrophic forD-glutamate over time, without the possibility of reversion to the wildtype phenotype.

Example 13 In Vivo Clearance of A. baumannii Δ0380/Δ3398 Strain AfterIntraperitoneal Administration in Mice

In order to evaluate the safety of A. baumannii Δ0380/Δ3398 as avaccine, bacterial counts were performed from the blood of BALB/c miceinoculated intraperitoneally (100 μL) with a 1× dose of the wild typeand Δ0380/Δ3398 mutant strains prepared in saline medium independentlyof each other. After 45 minutes and after 2, 4, 6, 12 and 18 hours ofthe administration, mice were euthanized and blood was obtained directlyfrom the heart and plated on LB agar with and without 10 mM D-glutamate,with D-glutamate for the auxotrophic strain and without for the wildtype.

We observed significant differences between the bacterial countsobtained with wild type and Δ0380/Δ3398 strains after 45 minutes postadministration (see FIG. 21). In the case of the Δ0380/Δ3398 mutantstrain, no colonies were recovered beyond 6 hours. These results suggestan acceptable threshold of security for the administration of thisstrain as a vaccine candidate, as this live attenuated bacterium iseliminated from the body within hours from its administration.

Example 14 Identification of Genes in P. aeruginosa PAO1 that EncodeGlutamate Racemases

Analysis of the genome sequence of P. aeruginosa strain PAO1 using theProtein Knowledgebase (UniProtKB) and the Pseudomonas Genome Databaserevealed a single putative glutamate racemase gene: PA4662, whichencodes a 265 amino acid protein. FIG. 4 compares and aligns thepredicted amino acid sequences for this putative glutamate racemase fromP. aeruginosa PAO1 with the two reported glutamate racemases from A.baumannii ATCC 17978 and the single reported glutamate racemase from E.coli K12. The Murl protein encoded by PA4662 of P. aeruginosa PAO1 has37.5% amino acid sequence similarity with the Murl protein encoded bythe E. coli K12 (MURI_ECOLI), 35.9% amino acid sequence similarity withthe Murl protein encoded by the A1S_0380 locus of A. baumannii(A3M1P5_ACIBT) and 37.5% similarity with the Murl protein encoded by theA1S_3398 locus (A3MA43_ACIBT) of A. baumannii.

Example 15 Construction and Characterization of a P. aeruginosaGlutamate Racemase Deficient Mutant

We used in vitro methods to construct a mutant allele, designatedΔPA4662, with an in-frame deletion corresponding to the coding region ofPA4662. This mutant allele was substituted for the corresponding wildtype allele in the chromosome of P. aeruginosa strain PAO1 by using thepEX18Gm allelic exchange system.

The plasmid pEX18Gm was used to generate an unmarked deletion in the P.aeruginosa PA4662 gene, LPA4662, by allelic exchange in PAO1 strain. ThepEX18GmUP/DOWNPA4662 plasmid was constructed by cloning two PCRfragments, approximately 1 kb in length, spanning the upstream anddownstream regions of the PA4662 of P. aeruginosa PAO1. The upstreamfragment was amplified using primers UPPA4662(HindIII)F_II andUPPA4662(NotI)R and the resulting PCR product was digested with HindIIIand NotI. The downstream fragment was amplified using DOWNPA4662(NotI)Fand DOWN0380(XbaI)R and the resulting PCR product was digested with NotIand XbaI. Digested upstream and downstream fragments were ligated intovector pEX18Gm linearized with HindIII and XbaI to generatepEX18GmUP/DOWNPA4662.

The pEX18GmUP/DOWNPA4662 plasmid was first introduced into E. coli S17-1by transformation. Briefly, electrocompetent E. coli S17-1 cells werecultivated with 15 μg/ml gentamicin overnight at 37° C. after applyingthe electric pulse. Following incubation, resulting colonies wereanalyzed by PCR using the primers UPPA4662(HindIII)F_II andDOWNPA4662(XbaI)R to confirm the desired presence of thepEX18GmUP/DOWNPA4662 plasmid.

The pEX18GmUP/DOWNPA4662 plasmid was introduced in P. aeruginosa PAO1strain by electroporation as described above and cells were cultivatedin LB with 30 μg/ml gentamicin for 3 days at 37° C. Independentlyisolated co-integrant colonies were inoculated into 1 mL of LB brothsupplemented with 10 mM D-glutamate and 15% sucrose and grown at 37° C.overnight while shaking. Cultures were then serially diluted using NaCl0.9% and dilutions were plated onto LB agar containing 15% sucrose and10 mM D-glutamate. Individual colonies of resolved co-integrants werepicked from LB agar plates containing 15% sucrose and 10 mM D-glutamateand inoculated in patches at comparable locations on LB agar plates withand without 10 mM D-glutamate. Resolvants with the APA4662 genotype grewonly on the LB agar with D-glutamate, and resolvants with the wild typegenotype grew on LB agar with or without D-glutamate.

In this P. aeruginosa strain, the resulting ΔPA4662 single mutantrequired exogenous D-glutamate for growth. FIG. 22 illustrates the patchtests that were used to distinguish individual resolved co-integrantswith the ΔPA4662 mutant genotype that grow only on LB agar containingD-glutamate from individual resolved co-integrants with the wild typegenotype that do not require D-glutamate for growth.

The presence of the appropriate wild type or in-frame deletion variantof PA4662 in the newly constructed mutants was confirmed by PCR usingprimers UPPA4662(HindIII)F_II/DOWNPA4662(XbaI)R; INTFWPA4662/INTRVPA4662and EXTFWPA4662/EXTRVPA4662 (screening using the last combination ofprimers is is illustrated in FIG. 23).

Our results showed that a single in-frame deletion at the PA4662 locusof P. aeruginosa PAO1 strain introduced by allelic exchange using thehighly homologous pEX18GmUP/DOWNPA4662 plasmid, conferred a stringentgrowth requirement for exogenous D-glutamate. This finding indicatesthat PA4662 is the only gene in P. aeruginosa PAO1 that directsproduction of functional glutamate racemase.

Example 16 Determination of the Lethal Doses (LD) of P. aeruginosa WildType and ΔPA4662 Glutamate Racemase Deficient Strain in a Mice Model ofAcute Infection. Evaluation of Antibody Immune Response (IgG) byIndirect ELISA

BALB/c mice (n=4 mice/group) were administered different doses of P.aeruginosa PAO1 wild type and ΔPA4662 with the purpose of determiningthe lethal doses of these strains during an acute sepsis infection.

For preparation of the administered inoculums, bacteria were grown in LB(wild type strain) and LB supplemented with 10 mM D-glutamate (ΔPA4662mutant) at 37° C. with shaking until an OD_(600 nm)=0.7 (1× dose).Cultures were then centrifuged and the bacterial pellet was washed 2times with LB. After cell washing, bacterial suspensions were adjustedwith NaCl 0.9% at different doses (0.1×; 0.4×; 1×; 4×; 10×y 40×),according to the previous OD_(600 nm) value, and inoculated (100 μL) inBALB/c mice by intraperitoneal injection (1× meaning as bacterialinoculums with OD_(600 nm)=0.7, 0.1× the bacterial inoculums diluted1:10, 2× the bacterial inoculums 2:1 concentrated, and so on . . . ).

Mice were monitored for 7 days after infection and survival rates weredetermined for different doses of injected bacteria. Lethal doses (LD)titer of each bacterial strain were determined considering the observedsurvival of mice in both cases, meaning LD₁₀₀ the minimal dose for which100% of susceptible mice will die.

In FIG. 24A we can see different degrees of survival in animals infectedwith increasing doses of P. aeruginosa PAO1 wild type strain. For thisstrain, the LD₁₀₀ is =0.4×. In FIG. 24B we can see different degrees ofsurvival in animals infected with increasing doses of the ΔPA4662mutant. For this strain, the LD₁₀₀ is >40×, a very high dose ofbacterial inoculum, which can lead to death of the mice from septicshock (and not due to replication of the bacteria). This indicates thatthis strain has a much reduced virulence (a dose 100 times higher thanthe wild strain LD₁₀₀ only decreases by 50% survival of mice).

In addition, we evaluated the antibody immune response (IgG) by indirectELISA. To that end, groups of 4 mice were immunized once with one of thefollowing doses: 0.1×, 0.4×, 1×, 4×, 10× and 40×. As shown in FIG. 44,one vaccine dose of 0.1× of ΔPA4662 mutant strain (1×=5×10⁸ CFU/mL) issufficient to trigger IgG production significantly (P<0.001), even whendetected at day 40 after the vaccine administration. Nonetheless,vaccine doses equal or greater than 0.4× elicit higher levels of IgGproduction.

As shown in FIG. 45, IgG levels are significantly incremented at day 7after administering the first vaccine dose (0.4×). However, antibodyproduction is significantly higher when the 2^(nd) vaccine dose isadministered (0.4×).

Lastly, ELISA was performed with respect to different strains of P.aeruginosa with sera obtained on day 34 from mice vaccinated with 3dosis (0.4×) of the ΔPA4662 strain (administrated on days 0, 14 and 28)and mice administrated saline (on the same days) to measure the capacityof ΔPA4662 vaccine to generate a broad immune response. Results similarto those observed with respect to P. aeruginosa strain PAO1 wereobtained with strain PA28562 whereas high levels of cross-reactivitywere also seen with respect to rest of P. aeruginosa strains tested(PA51430664, PA26132, PAST175, PA29475 and PA12142) (FIG. 46). Thisdemonstrates that immunization with strain ΔPA4662 not only generatesantibodies against the isogenic wild type strain, but also generates IgGantibodies that react against multiple P. aeruginosa strains.

Example 17 Morphological Analysis of P. aeruginosa PAO1 Wild Type andΔPA4662 Mutant Strains by Electron Microscopy

For obtaining electron micrographs by transmission electron microscopy(TEM), the ΔPA4662 mutant strain was first grown in LB agar supplementedwith 10 mM D-glutamate for 18 h at 37° C. and finally plated onto MHagar, LB and LB supplemented with MgCl₂ (30 mg/L) and CaCl₂ (75 mg/L)and incubated for 18 h at 37° C. After incubation, 2-3 colonies weredissolved in PBS buffer, the suspension was centrifuged, and theresulting pellet was first washed with cacodilate buffer and immediatelycells were fixed in 2.5% ice cold gluteraldehyde prepared in sodiumcacodilate buffer (0.2 M, pH 7.4) for 4 hours at room temperature. Thepellets were then washed with cacodilate buffer, dehydrated in acetoneand embedded in Spurr (Spurr's Epoxy Embedding Medium). Ultrathinsections (70 nm) of the samples were stained with uranyl acetate andplumb citrate for observation in a JEOL JEM 1010 (80 kV) transmissionelectron microscope.

FIG. 25 shows the different stages of the cell wall degeneration of theΔPA4662 mutant strain: from cells with altered conformation that losetheir rigid structure to cells that present several ruptures anddisplacement of the outer membrane, lysis and extrusion of theintracellular content (especially genetic material). The mechanism ofbacterial destruction can be followed with this order: 1) the absence ofthe cell wall leaves the bacterial protoplasm surrounded only by theinner cell membrane (protoplast), which makes this cell body totallyexposed to variations in the tonicity of the medium; 2) protoplastsburst and leave traces of the cytoplasmatic membranes—called “ghosts”that can aggregate, also individual membranes and liposomes can bevisible.

Example 18 Protection of BALB/c Mice Against Challenge with P.aeruginosa PAO1 Strain by Immunization with the ΔPA4662 Mutant

To evaluate the efficacy of the ΔPA4662 strain as a vaccine, BALB/c mice(n=8) were administered 100 μL of the ΔPA4662 strain (0.4× dose insaline) on days 0 and 14. Control mice were administered only salineidentically at days 0 and 14. Twenty five days after the secondinjection, mice were challenged with P. aeruginosa PAO1 wild type strain(0.4× dose in saline) in order to establish a lethal systemic infectionin both cases (100 μL of intraperitoneal injection). After thechallenge, mice were monitored for 7 days to determine the survival rateof vaccinated mice compared to control mice (unvaccinated).

When infected with a 0.4× dose of the P. aeruginosa PAO1 wild typestrain, 8 deaths were observed in the group of unvaccinated mice duringthe first 15 hours, which means a mortality rate of 100% in this group(n=8). In contrast, all vaccinated mice (n=8) survived to the challenge,overcoming the infection, which means a 100% survival (see FIG. 26A)rate in this group. Differences in survival between the two groups wereextremely statistical significant (P<0.0001, according to the Mantel-Coxlog-rank test).

These results suggest that vaccination with the APA4662 strain canprovide protective immunity against infection with P. aeruginosa.

These results are further confirmed as follows. First, FIG. 26B showsthe percent survival (87.5% vaccine efficacy) of BALB/c mice (n=8)following intraperitoneal infection with a 0.4× dose of P. aeruginosaPAO1 wild type strain. Vaccinated mice were immunized on days 0 and 14with P. aeruginosa ΔPA4662 strain (0.04× dose) and infected with thewild type strain at day 25. Non-vaccinated mice were administered salineon days 0 and 14 and infected with the wild type strain at the same day.*P<0.0001 survival of vaccinated group compared to unvaccinated group.P-value, according to the Mantel-Cox test (log-rank test). Secondly,FIG. 47 shows that vaccinated mice had a significant decrease in CFUs ofP. aeruginosa in liver, spleen and lungs after 10 hours of acute sepsisinfection.

To evaluate the protective efficacy of the ΔPA4662 strain as a vaccineusing one of the preferred route for administration in humans, BALB/cmice (n=8/per group) were inyected with 100 μL of the ΔPA4662 strain(0.4× dose in saline) on days 0, 14 and 28 using the intramuscularroute. Control mice were administered only saline identically at days 0,14 and 28. At day 34, blood were collected from the submandibular veinfrom all mice without euthanasia, the sera was separated and IgGquantification was performed as above. Significant differences betweenIgG antibody production were observed between the group of miceimmunized compared to control group (P<0.0001, according to unpaired ttest) (FIG. 49).

At day 35, mice were challenged with P. aeruginosa PAO1 wild type strain(0.4× dose in saline) in order to establish a lethal systemic infectionin both cases (100 μL of intraperitoneal injection). After thechallenge, mice were monitored for 7 days to determine the survival rateof vaccinated mice compared to control mice (unvaccinated). After thechallenge, 8 deaths were observed in the group of unvaccinated micewhich means a mortality rate of 100% in this group. In contrast, allvaccinated mice survived to this challenge, overcoming the infection.This means a 100% survival rate in this group (see FIG. 50). Differencesin survival between vaccinated and control mice were extremelystatistical sifnificant (P<0.0001, according to the Mantel-Cox log-ranktest). This result suggests that vaccination using the intramuscularroute of administration is as effective as the intraperitoneal route.

Example 19 Passive Immunization with ΔPA4662 Vaccine Antisera

In situations in which the completion of a vaccination schedule prior tobacterial exposure is not possible, when individuals cannot synthesizeantibody, or even after exposure to the pathogen, the use of passiveimmunization may be beneficial. Those antibodies formed with ΔPA4662vaccination can be removed from the host and transferred into anotherrecipient where they can provide immediate passive immunity or helpfight the infectious disease.

We first determined if vaccine serum from mice immunized with theΔPA4662 strain could be used to passively immunize mice before theexposure to P. aeruginosa. One dosis of vaccine or naïve sera wereadministered by intraperitoneal injection (200 μL) to BALB/c mice (n=8)3.5 h prior to infection with 0.4× dose of P. aeruginosa PAO1 wild typestrain and survival of mice was monitored for 3 days after thechallenge. As shown in FIG. 27A, all mice administrated vaccine serumwere protected from disease, whereas 50% of the mice receiving naïveserum succumbed to infection (P=0.025; Mantel-Cox test (log-rank test)).

These results demonstrate that passive immunization with serum from micevaccinated with the ΔPA4662 strain is able to confer a significant levelof protection from infection, when administered prior to infection,whereas antibodies alone are sufficient for providing protectiveimmunity against P. aeruginosa PAO1 acute sepsis.

Next, we determined if vaccine serum from mice immunized with theΔPA4662 strain could be given as a medication to nonimmune mice havingalready received a lethal injection of P. aeruginosa, in order toameliorate the prognostics of the disease. As a treatment, two dosis ofvaccine or naïve sera (150-200 μL) were given via intravenous injectionto BALB/c mice (n=7) 2 h and 4.5 h after the infection with a 0.4× dosisof P. aeruginosa PAO1 wild type strain. Survival of mice was monitoredfor 2 days. Of note, when the first treatment with sera wasadministrated, all mice presented visible symptoms of an acute sepsisinfection. As shown in FIG. 27B, all mice receiving the lethal dosis ofP. aeruginosa succumbed to infection, however, mice administratedvaccine serum survived significantly longer (P=0.0112; Mantel-Cox test(log-rank test)) than mice receiving naïve serum. Thus, vaccine seraoffered mice life maintenance during several hours after the developmentof the acute lethal disease and helped protect right away. In regards tocorrelates of protection and treatment with vaccine sera, these resultsalso suggest that the optimal antibodies titres and administrationschedule may need to be determined.

Example 20 Environment Safety Assessment of the ΔPA4662Strain—Evaluation of Water Osmolisis

A vaccine candidate should also be construed to mean one bacterium whichis incapable of replicate and to persist in the general environment,once it leaves the vaccinated individual. To compare the ability of P.aeruginosa PAO1 wild type and ΔPA4662 mutant strain to be long-timetraced in the general environment, we evaluated survival of thesestrains in water without any contribution of nutrients or salts, at 37°C. and agitation (180 rpm) for the time necessary to observe the loss ofviability by cellular osmolisis.

Daily samples of culture were taken initially for 3 days, next, sampleswere taken twice a week until day 62, and finally, at least once everytwo weeks for the determination of CFU counts in LB agar (wild typestrain) and LB agar supplemented with 10 mM D-glutamate (mutant strain).All cultures were performed in triplicate.

A significant decrease in the viability of the ΔPA4662 strain was seen,and no viable bacteria were recovered within and after 17 days ofculture. In contrast, its wild counterpart, the wild type strain,remained widely recoverable after the 25^(th) day of culture (see FIG.28) and survived until day 143.

Example 21 Evaluation of the Stability of the Auxotrophic Phenotype inthe ΔPA4662 Strain

To test the irreversibility of the nutritional auxotrophy of P.aeruginosa ΔPA4662 for the compound D-glutamate, ΔPA4662 strain wasgrown in 100 mL of LB supplemented with 10 mM D-glutamate in optimalconditions for 5 days at 37° C. with agitation (180 rpm). Samples fromthis culture were taken at the beginning of incubation and at days 3 and5 for determination of CFU in LB agar and LB agar supplemented with 10mM D-glutamate. All cultures were performed in triplicate. In thehypothetical case of a phenotype reversion, similar bacterial countsshould be recovered in agar plates over time, independently of thepresence or absence of the compound in the medium. In contrast, weobserved significant differences between the bacterial counts obtainedwhen the culture was plated onto agar medium with and withoutD-glutamate.

Resulting bacterial counts were significantly higher in the first case(agar plates supplemented with D-glutamate), at the initial stage ofincubation (0 days) and on days 3 and 5 (see FIG. 29) (P=0.0059,according to Student's t test). The recovery of a significantly lowernumber of colonies in the agar plates without D-glutamate can be due toa residual growth derived from the accumulation of this compound in thecytoplasm of bacterial cells during growth in supplemented media. Thisdifference indicates that ΔPA4662 strain remains auxotrophic forD-glutamate over time, without the possibility of reversion to the wildtype phenotype.

Moreover, in FIG. 51 we observed significant differences between thebacterial counts obtained with wild type and ΔPA4662 strains after 1hour after intraperitoneal administration. In the case of the ΔPA4662mutant strain, no colonies were recovered beyond 1 hour. These resultssuggest an acceptable threshold of security for the administration ofthis strain as a vaccine candidate, as this live attenuated bacterium iseliminated from the blood within a few hours from its administration.

Example 22 Construction and Characterization of Single and Double MutantStrains of S. aureus without Glutamate Racemase and/or D-amino AcidTransaminase

Homologous double recombination was carried out using the temperaturesensitive replication vector pMAD to construct the mutant strains.First, the unmarked deletion of the annotated glutamate racemase (murl)and D-amino acid transaminase (dat) genes were achieved independently.Each single mutant strain was called Δmurl and Δdat, respectively.

To achieve the construction of these two mutants, fragments of 1000 bpthat correspond to the upstream (left) and downstream (right) DNA thatflanked the genes were amplified by PCR and cloned separately into theshuttle plasmid pMAD. The resulting recombinant plasmids were used toremove the chromosomal murl and dat genes located on the chromosome ofS. aureus 132 wild type strain.

S. aureus 132 is a clinical MRSA strain used in the present invention asa model organism of the species “Staphylococcus aureus” to generateauxotrophic mutants to D-glutamate. This is a clinical strain resistantto methicillin (MRSA) (Vergara-Irigaray et al, Infection and Immunity,77 (9): 3978-3991 (2009)).

The upstream fragment of the murl gene was obtained by PCR amplificationusing the combination of murIUP(Mlul)F and murlUP(NotI)R primer pair,and subsequently digested by Mlui and NotI restriction enzymes. Thedownstream fragment of the murl gene was obtained by PCR amplificationusing the murIDOWN(NotI)F and murlDOWN(BgIII)R primers followed bydigestion with NotI and BgIII enzymes. The digested upstream anddownstream fragments of the murl gene were cloned into the pMAD vectorpreviously linearized with Mlul and BgIII enzymes, yielding theconstruction named pMAD_UP/DOWN_murl.

The same strategy was completed for the construction of the plasmidpMAD_UP/DOWN_dat. In this case, the upstream and downstream fragments ofthe dat gene were amplified using the primer pairsdatUP(Mlul)F/datUP(NotI)R and datDOWN(NotI)F/datDOWN(BgIII)R,respectively.

pMAD_UP/DOWN_murl and pMAD_UP/DOWN_dat were introduced into E. coli TG1by electroporation. Transformant colonies were selected on LB agarsupplemented with ampicillin (100 μg/mL) plus X-Gal (150 μg/mL). Afterincubation at 37° C. for 18 h, ampicillin-resistant blue colonies werePCR checked for the presence of pMAD_UP/DOWN_murl or pMAD_UP/DOWN_datwith the primer combinations murlExtF/murlExtR or datExtF/datExtR,respectively.

The constructions pMAD_UP/DOWN_murl and pMAD_UP/DOWN_dat extracted fromE. coli TG1 cells were independently introduced into the intermediatecloning strain S. aureus RN4220 previous electroporation into thetargeted strain S. aureus 132. The selection of S. aureus RN4220colonies that contained recombinant plasmids were is performed on TSBagar supplemented with erythromycin (10 μg/mL) plus X-Gal (150 μg/mL)after incubation at 30° C. for 24-48 h.

Each recombinant plasmid was extracted from S. aureus RN4220 andindependently introduced into S. aureus 132 wild type strain byelectroporation. As well, erythromycin-resistant blue colonies of S.aureus 132 that harboured each construction were grown on TSB agar witherythromycin (10 μg/mL) plus X-Gal (150 μg/mL) at 30° C. for 24-48 h.

To delete the chromosomal murl gene of S. aureus 132 wild type strain,one colony of S. aureus 132 wild type harbouring pMAD_UP/DOWN_murl wastransferred to a 5 mL of TSB with 10 μg/mL of erythromycin and grown at30° C. for 2 h. Afterwards, the culture were incubated at 43.5° C., anon-permissive temperature for pMAD replication, leading the integrationof pMAD_UP/DOWN_murl into the bacterial chromosome via a singlecrossover recombination upstream or downstream region of murl gene.After 6 hours, the TSB culture was serially diluted and spread onto TSAplates supplemented with erythromycin (10 μg/mL) plus X-Gal (150 μg/mL)and incubated at 43.5° C. for 18 h. Several of the resulting colonieswere transferred to a 5 mL of TSB without antibiotic and incubated for18 h at 30° C. in order to induce a second crossover event that lead topMAD_UP/DOWN_murl excision from the chromosome. The selection of whitecolonies, which no longer contain pMAD_UP/DOWN_murl, was carried out byplating serial dilutions on TSB plates with X-Gal (150 μg/mL). Eachselected colony was transferred onto TSA with X-Gal (150 μg/mL) as wellX-Gal (150 μg/mL) plus erythromycin (10 μg/mL). Erythromycin-sensitivewhite colonies were checked by PCR for the deletion of murl gene (Amurl)using murlExtF/murlExtR, muriseqF/murlseqR and murlF/murlR primer pairs.

The deletion of dat gene from the chromosome of S. aureus 132 wild typestrain was performed following the protocol previously described formurl but using the pMAD_UP/DOWN_dat plasmid. In this case, coloniessuspected to no longer contain the dat gene (Δdat) were confirmed usingdatExtF/datExtR, datseqF/datseqR and datF/datR primer combinations.

To generate the double mutant Δmurl/Δdat the pMAD_UP/DOWN_dat plasmidwas introduced by electroporation into the mutant Δmurl strain and thesame protocol described before for the single mutants was performed. Inthis case, the recovering of double mutants required adding exogenousD-glutamate to the medium (20 or 10 mM for TSB or TSB agar respectively)since colonies with double gene deletion require this D-aminoacid forgrowth. The absence of the muri and dat loci in the genome of S. aureus132 wild type strain was confirmed by PCR using the following primers:murl ExtF/murl ExtR, murlseqF/murlseqR, murlF/murlR, datExtF/datExtR,datseqF/datseqR and datF/datR.

The culture of the different mutant strains in medium with and withoutD-glutamate revealed that the single deletion of murl or dat genes doesnot affect bacterial growth. However, the double mutant requires thepresence of D-glutamate in the medium for growth. FIG. 30 represents thecolony screening method carried out for the selectederythromycin-sensitive white colonies resulting from the secondcrossover event of the single mutant Δmurl.

FIG. 31 shows PCR confirmation of the different deletions in the threemutant strains of S. aureus 132 wild type. The results obtained so fardemonstrate that the presence of either any of the two wild type loci,murl or/and dat, is sufficient for the normal growth of S. aureus 132 inTSB agar without added D-glutamate, and that the simultaneous deletionof both genes make this strain unable to grow without the presence ofD-glutamate. In conclusion, it is demonstrated that the murl and datgenes of S. aureus 132, are the only genes involved in the biosynthesisof D-glutamate in this strain.

It is thus worth noting that the inactivation of both glutamate racemaseand D-amino acid transaminase enzymes results in an auxotrophy toD-glutamate and that the method for obtaining auxotrophic mutantspertaining to Staphylococcus aureus species is independent of the strainselected.

Example 23 Effect of D-glutamate on the Double Mutant Δmurl/Δdat Growthand Viability in Liquid Culture Medium

To evaluate the growth and viability curve of the S. aureus doublemutant Δmurl/Δdat in comparison with the S. aureus 132 wild type, bothstrains were inoculated in exponential growth phase to a final 1/200dilution into 100 ml of TSB and TSB supplemented with 20 mM D-glutamate,and incubated at 37° C. with constant shaking (180 rpm). At 1, 2, 3, 4,5 and 18h, the optical density of the culture and bacterial isconcentration were determined. The optical density was assessed bymeasuring aliquots of each flask at OD_(600 nm) whilst the bacterialconcentration (CFU/mL) was calculated by spreading serial 10-folddilutions onto TSB agar plates. All the cultures were performed intriplicate.

Growth curves for the S. aureus wild type strain as well as for doublemutant strain Δmurl/Δdat were performed to evaluate the effect of theabsence of D-glutamate in the medium along the time, as well as, theviability of the strains in the presence and absence of this compound.

A total absence of growth was observed for the double mutant strain inTSB medium without D-glutamate (FIG. 32A), being almost totally restoredwhen adding D-glutamate to the medium. With regard to bacterialviability (FIG. 32B) it is shown that the viability of the double mutantsignificantly decreased (2 Log₁₀) at 18 h due to the limitation ofD-glutamate in the culture medium.

Example 24 Morphological Analysis of S. aureus 132 Wild Type and DoubleMutant Δmurl/Δdat Strains by Electron Microscopy

To prepare electron microscopy samples pellets recovered from the liquidcultures of S. aureus wild type and double mutant Δmurl/Δdat strains inTSB supplemented with 20 mM of D-glutamate (37° C. for 16 h) were washedtwice in saline and re-suspended in 1 mL of TSB. 50 μL of eachsuspension was transferred to 5 mL of TSB supplemented with increasingconcentrations of D-glutamate: 0, 0.1, 1.5 and 20 mM and incubated at37° C. for 3 hours with constant shaking (180 rpm). The obtainedbacterial cultures were centrifuged and pellets were washed twice withPBS. Afterwards, pellets were fixed with paraformaldehyde 4% in PBS 1MpH 7.4 for 30 minutes at room temperature and shaking. The samples wereadditional washed twice with PBS and dehydrated with a series ofincreasing alcohol concentrations (50%, 70%, 90%, 100%) for 15 minuteseach. Finally, the samples were dried at critical point with CO₂(Bal-Tec CPD 030). One drop of each sample was placed onto a slide coverand fixed onto an aluminium support for gold coating (Bal-Tec SCD 004sputter coater). Samples were observed and photographed in atransmission electron microscope Jeol JSM-6400.

Microscopic observation enabled to detect substantial morphological andstructural changes in the double mutant Δmurl/Δdat strain as D-glutamatein the medium decrease. FIG. 33 shows the scanning electron imagescomparing wild type and double mutant strains after growing in TSBmedium supplemented with different concentrations of D-glutamate.

FIG. 33A shows that the double mutant is unable to divide withoutexternal addition of D-glutamate. Accordingly, at 0 mM of D-glutamatethe bacterial cells detected mostly correspond to the initial inoculumwhich was previously grown in the presence of this compound. Someabnormal cellular shapes can be also observed as consequence ofincomplete cellular division occurred due to reminiscent intracellularD-glutamate. When the double mutant is incubated in medium supplementedwith 0.1 mM of D-glutamate some cellular division occurred but continuesbeing atypical owing to the low concentration of this D-amino acid.Thus, the process of cell wall biosynthesis and division is not completethus generating deformed cells and protoplasts (bacterial cells lackingpeptidoglycan but still harbouring plasmatic membrane). In the presenceof D-glutamate at 1.5 mM the bacterial density slightly increased inrelation to cultures derived from medium with lower concentrations ofthis compound, indicating a higher growth rate of the double mutant.Protoplasts and lengthened shapes are still present but typicalspherical bacterial cells with the same configuration than the wild typestrain can be observed. Finally, when the medium is supplemented withD-glutamate 20 mM the cellular density as well as morphology of thedouble mutant is comparable with that shown by the wild type strain. Noatypical division pattern is observed.

On the other hand, FIG. 33B displays different scanning electronmicrographs of the bacterial cultures of the double mutant and wild typestrains incubated in medium supplemented with 0.1 mM of D-glutamate.Wild type strain shows the characteristic spherical shape ofgram-positive coccus, high cellular density and a typical divisionpattern. S. aureus division is characterized by an incomplete cell wallseparation thus appearing bacterial cells in grape-clusters that remainconnected by segments of peptidoglycan. Conversely, cells of the doublemutant become forming part of large compact aggregates or conglomeratesas consequence of the exacerbated incomplete cell division. The lack ofenough concentration of D-glutamate in the medium impaired peptidoglycanbiosynthesis and correct cell wall division. Indeed, cellular density islower than that observed in the wild type preparations. Moreover,abundant deformed shapes are visualized including unusual lengthenedbacteria. Under this condition, the double mutant also shows rough andirregular surface in comparison with the wild type strain.

Furthermore, the progressive disintegration of the cell wall, bacterialdegeneration and subsequent bacterial death was detected in the doublemutant by transmission electronic microscopy. To this end, samplepreparations were obtained by short-term maintenance of the doublemutant strain in absence of D-glutamate as described in example 4. Ascan be seen from FIG. 34, this approach allowed to the observation ofdifferent gradual disintegration stages until bacterial death. Initialmorphological changes observed included diminishing of murein layer andconsequently an increase in cell size (protoplasts), since peptidoglycanconstitute the mechanically resistant part of the wall cell.Permeability and disruption of the plasma membrane can be also observedwhich results in the extrusion of cytoplasmic constituents. Large amountof debris, membrane aggregates, liposomes and genetic material can beseen around these collapsed cells. On the other hand, some bacterialcells still maintain an intact envelope and hence the typical S. aureusbacterial shape and size could be observed.

Example 25 Determining the Lethal Dose (LD) of S. aureus Wild Type 132and Double Mutant Δmurl/Δdat Strain in BALB/c Mice in an Acute InfectionModel

With the aim of producing a systemic infection in BALB/c mice, salineinoculum with 3% of hog mucin of S. aureus wild type and double mutantstrains were intraperitoneally administered to mice.

For preparation of the inoculum, bacteria were grown in TSB (wild type)and TSB supplemented with 20 mM D-glutamate (double mutant) at 37° C.with shaking (180 rpm) until reaching OD_(600 nm) of 0.7. The cultureswere centrifuged, washed twice with saline serum, and re-suspended insaline serum with 3% of hog mucin (wild type and double mutant strainsfor intraperitoneal sepsis model) or in saline serum (double mutant forimmunize animals, following examples) to a final concentration of ˜10⁸CFU/mL. This suspension is called 1× and corresponds to 5×10⁷ CFU/mousewhen a volume of 250 μL is administered to mice. Bacterial suspensionswere further adjusted at different doses (for instance, a suspension 3×is understood as the bacterial inoculum three-times concentrated and soon).BALB/c (n=3-4) were inoculated intraperitoneally with differentdoses (250 μL) of bacterial suspension and monitored for 14 dayspost-infection. LD₁₀₀ is defined as the minimum lethal dose for 100%mice mortality.

In FIG. 35A is shown different degrees of survival in mice whenadministered increasing doses of S. aureus wild type strain. The minimumdose that reduces survival of the mice to 0% was determined as 3×. InFIG. 35B survival in mice when administered increasing doses of doublemutant strain is illustrated. In clear contrast this figure shows thatinoculating a dose of the double mutant 10-fold higher than the LD100 ofthe wild type strain results in a 100% survival rate. Therefore, thelethal dose for the double mutant is greater than 30× LD₁₀₀>30×. Thisclearly demonstrates that the double mutant of S. aureus is a highlyattenuated strain showing lower virulence potential than the wild typecounterpart strain.

Example 26 Determining the Bacterial Load in Spleen and Blood of BALB/cMice Pre-Immunized with the Double Mutant Δmurl/Δdat Using a SystemicInfection Model

To evaluate the effectiveness (protection level) of the S. aureus doublemutant Δmurl/Δdat strain as a vaccine, two independent experiments wereperformed.

Firstly, BALB/c mice (n=4-6) were intraperitoneally pre-immunized (250μL) with the double mutant Δmurl/Δdat strain in saline serum (10× dose)on days 0 and 14. One group of mice were identically administered 250 μLsaline at days 0 and 14. At day 21, mice were infected intraperitoneallywith a lethal inoculum (5× dosis, 250 μL with 3% of hog mucin) of S.aureus 132 wild type strain. At 20 hours post-infection mice wereeuthanized with sodium thiopental. The spleen of each mouse wasaseptically removed and after being homogenized in saline, the CFUs pergram of organ were determined by plating serial dilutions in TSB agar.The presence of bacteria in blood was evaluated by inoculating 50 μL ofblood sample aseptically removed from the mice heart into 5 mL of TSBmedium. The bacterial inoculum were prepared and adjusted as describedpreviously.

The protective effect of the vaccination with the double mutantΔmurl/Δdat was confirmed when it was observed that pre-immunization withthis strain causes a significant reduction in bacterial load in spleensof mice infected with a lethal dose of S. aureus 132 wild type strain.Indeed, we observed a severe reduction (2 Log₁₀) in the bacterial loadof immunized mice compared to non-immunized mice (P=0.0095, Mann-WhitneyU-test, FIG. 36A). In addition, the absence of bacteria in the blood ofall vaccinated mice (negative blood cultures) further supports theprotective effect of vaccination (FIG. 36B).

These results are further confirmed as follows. First, FIG. 37 showsthat in pre-immunized BALB/c mice (n=8-9) with the double mutantAmurl/Adat strain (8× dose in this case) the bacterial counts in spleen(FIG. 37A) and blood (FIG. 37B) were significantly lower compared to thenon-immunized group (P=0.0006 and P=0.0002, respectively, according toMann-Whitney U test) 22 hours after infected with a lethal inoculum ofS. aureus 132 wild type strain (5× dose, 250 μL with 3% of hog mucin).Immunization schedule was performed as above.

Example 27 Protection of BALB/c Mice Against Challenge with S. aureus132 Strain by Immunization with the Δmurl/Δdat Mutant

To evaluate the efficacy of the Δmurl/Δdat as a vaccine, BALB/c mice(n=10-13) were administered 250 μL of the Δmurl/Δdat strain (10× dose insaline) on days 0 and 14. Control mice were administered only salineidentically at days 0 and 14. Seven days after the second injection,mice were challenged with a lethal dose of S. aureus 132 wild typestrain (5× dose in saline with 3% hog mucin) in order to establish alethal systemic infection in both cases. After the challenge, mice weremonitored to determine the survival rate of vaccinated mice comparedwith control group (non-vaccinated).

When infected with a 5× dose of the S. aureus 132 wild type strain, 9deaths were observed in the group of non-vaccinated mice, which means amortality rate of 90% in this group (n=10). In contrast, 8 mice ofvaccinated group (n=13) survived to the challenge, overcoming theinfection, which means a 61.5% survival rate in this group (see FIG.38). Differences in survival between the two groups were statisticalsignificant (P<0.031, according to Mann-Whitney U test).

These results show that vaccination with the Δmurl/Δdat strain canprovide protective immunity against subsequent infection with S. aureus.

Example 28 Quantification of IgG Antibodies Against the Isogenic S.aureus 132 Δspa Strain Through Indirect ELISA in BALB/c Mice Subjectedto Vaccination the Double Mutant Δmurl/Δdat Strain

To evaluate the immune response to vaccination mediated by antibodies,BALB/c mice (n=10) were immunized by intraperitoneal injection (250 μL)of double mutant Δmurl/Δdat in saline (10× dose) on days 0 and 14. Atday 21, mice were anesthetized with sodium thiopental and blood wascollected via retro-orbital plexus puncture. Sera were separated fromblood cells by centrifugation and stored at −80° C. until analysis.

IgGs detection was performed using an indirect enzyme linkedimmunosorbent assay (ELISA). 96-well ELISA plates were coated with wholeS. aureus 132 Δspa strain. This strain is an isogenic strain of S.aureus 132 wild type strain defective for Protein A. Thus, thewhole-bacteria was fixed to the bottom of the wells after 18 h ofincubation at 4° C. in carbonate-bicarbonate buffer 100 mM, pH 9.6 (1/10 dilution of a culture with OD₆₀₀=1). Five (5) washes were performedwith phosphate buffered saline solution (PBS) buffer to remove unfixedbacteria. Blocking residual sites was performed in two steps to reducenon-specific interactions with the mouse sera. Firstly, plates wereincubated at room temperature for 1 h with 100 μL per well of blockingsolution (5% skim milk in PBS) and secondly, at 37° C. for 1 h with 100μL of rabbit serum ( 1/1000). After 5 washing steps with wash buffer(0.005% Tween 20 in PBS), plates were incubated overnight at 4° C. with100 μL of serial diluted sera in dilution buffer (DMEM medium with 5 to10% FCS). The following day, 5 washes were performed with wash buffer toremove unreacted antibodies and 100 μL of secondary antibody was addedper well (anti-mouse IgG peroxidase HRP-labeled) diluted 1/5000 indilution buffer. Incubation was performed over 1 h at room temperature.Plates were washed 5 times with wash buffer. To perform the developprocess the plates were incubated for 3 min with 100 μl of TMB(HRP-peroxidase substrate). The reaction was stopped with 50 μL of 1 MHCl per well. Colorimetric measure was performed at 450 nm. A positive(anti-Staphylococcus aureus monoclonal antibody), negative (serum fromnon-vaccinated mice) and reference (dilution buffer) controls wereincluded in all plates. To determine the titers of IgGs for each serum,the endpoint titer was estimated. Titers were defined as the last serumdilution with an absorbance 0.1 point higher than the reference control(dilution buffer).

Thus, the blood samples collected from each mouse were used to determinethe titer of antibodies (IgG) against S. aureus 132 Δspa strain byELISA, hence measuring the ability of the vaccine to generate an immuneresponse. Significant differences between IgG antibody production wereobserved between the group of mice immunized with a 10× dose of themutant Δmurl/Δdat compared with mice in the control group (P<0.0001,according to Mann-Whitney U test) (FIG. 39), demonstrating the efficacyof this strain in triggering IgG responses in mice.

Example 29 Cross-Reactivity of IgG Antibodies Generated with the DoubleMutant Δmurl/Δdat Against Unrelated S. aureus Strains

ELISA was performed with the sera indicated in the example 28 withrespect to USA300LAC, RF122, ED133 and ED98 strains to evaluate theantibody-mediated immune response in BALB/c mice immunized with theΔmurl/Δdat strain against unrelated S. aureus strains from differentorigin and thus, measure the ability of the vaccine to generate a broadimmune response. It is well known that S. aureus USA300LAC is anepidemic MRSA strain cause unusually invasive disease in healthyindividuals being a predominant cause of community acquired infectionsin United States, Canada and Europe. On the other hand, RF122 (bovine,ST151 and CC151), ED133 (ovine, ST133 and CC133) and ED98 (poultry, ST5and CC5) were selected as representative strains of three major clonesof animal host-adapted S. aureus strains that cause pathogenesis inlivestock.

To that end, plates were processed as described in example 28 but werepreviously “coated” with each of the above strains, independently.

As shown in FIG. 40, high significant titers of antibodies were detectedagainst the four unrelated S. aureus strains demonstrating thatimmunization with Δmurl/Δdat strain not only generates antibodiesagainst the isogenic 132 Δspa strain, but also against the relevantclinical strain USA300LAC, as well as three other strains well-adaptedto animal hosts.

Example 30 Environmental Safety Assessment of the Δmurl/ΔdatStrain—Evaluation of Water Osmolisis and Resistance to DesiccationConditions

The live attenuated vaccine candidate should be unable of persisting inthe general environment once it leaves the vaccinated individual withthe aim of keeping to a minimum the associated risks.

To compare the ability of S. aureus wild type and Δmurl/Δdat strains tobe long-time traced in the general environment, we evaluated survival ofthese strains in water without any contribution of nutrients or salts,at room temperature and under agitation (180 rpm) conditions for thetime necessary to observe the loss of viability by cellular osmolisis.Daily samples of the suspension were taken for the determination of CFUcounts in TSB agar (wild type strain) and TSB agar supplemented with 10mM D-glutamate (mutant strain). All cultures were performed intriplicate.

As shown in FIG. 41 a decrease in the viability of the mutant Δmurl/Δdatstrain was observed along the time being the time elapse for a 2-Log₁₀reduction 24 hours. Moreover, no viable bacteria of Amurl/Adat strainwere recovered beyond 72 hours of culture.

In addition, FIG. 52 shows the resistance of double mutant Δmurl/Δdatstrain to dryness compared to the wild type strain. To evaluate droughtresistance the cell viability of the wild type and double mutant strainswas tested by spotting dilution series of washed cells in theexponentially growth phase into nitrocellulose filters (0.45 μm poresize). The filters were either not dried (growth control) or dried for12 or 18 hours inside a sterile petri plate at 37° C. (drought stressconditions) before they were placed on TSB plates supplemented withD-glutamate and incubated for 24 hours at 37° C. All cultures anddilution series were performed in triplicate.

As shown in the figure, no difference in the cell viability was observedfor the wild type strain after keep under drought conditions comparedwith growth control (not dried filters). In contrast, the growth of the2-Log₁₀ diluted culture of the Δmurl/Δdat strain notably decreased after12 hours of dryness. Complete absence of cell viability (no grown) wasobserved in the same dilution of the double mutant when keep underdesiccation stress for 18 hours. These results indicate that theΔmurl/Δdat strain is more sensitive to desiccation than the wild typeparent strain and further support an appropriate threshold of securityfor its use as a vaccine.

Example 31 Evaluation of the Stability of the Auxotrophic Phenotype inthe S. aureus Δmurl/Δdat Strain

To test the irreversibility of the nutritional auxotrophy of S. aureusΔmurl/Δdat for the compound D-glutamate, Amurl/Adat strain was grown in100 mL of TSB supplemented with 20 mM D-glutamate in optimal conditionsfor 11 days at 37° C. with agitation (180 rpm). Samples from thisculture were taken at the beginning of incubation and at days 3, 5 and11 for determination of CFU in TSB agar and TSB agar supplemented with10 mM D-glutamate. All cultures were performed in triplicate. In thehypothetical case of a phenotype reversion, similar bacterial countsshould be recovered in agar plates over time, independently of thepresence or absence of the compound in the medium. In contrast, weobserved significant differences between the bacterial counts obtainedwhen the culture was plated onto agar medium with and withoutD-glutamate.

Resulting bacterial counts were significantly higher in the first case(agar plates supplemented with D-glutamate), at the initial stage ofincubation (0 days) and on days 3, 5 and 11 (see FIG. 42). Thisdifference indicates that Δmurl/Δdat strain remains auxotrophic forD-glutamate over time, without the possibility of reversion to the wildtype phenotype.

Example 32 In Vivo Clearance of S. aureus Δmurl/Δdat Strain AfterIntraperitoneal Administration in Mice

To evaluate the security of the S. aureus Δmurl/Δdat strain wheninoculated in the organism to be used as a vaccine, bacterial loads weredetermined in spleen, kidney and blood of BALB/c mice inoculated withΔmurl/Δdat strain compared to wild type strain. Mice (n=3/per strain)were intraperitoneally administrated (250 μL) with a sub-lethal 0.7×dose of wild type strain and with 10× (13-times higher dose) ofΔmurl/Δdat strain. Both cultures were prepared in saline with 3% of hogmucin independently of each other as previously described. One mouse pergroup was euthanized with sodium thiopental at post-infection days 1, 2and 6 to determine the bacterial load in organs and blood. Thus, spleenand kidney were aseptically processed as described above, and CFU pergram of organ were determined by plating serial dilutions in TSB agar(wild type) and TSB agar supplemented with 10 mM D-glutamate (doublemutant). Bacterial load in blood was determined by plating 50 μl ofblood aseptically removed from from heart.

In the acute sepsis model, the infection occurs with a rapid spread ofthe bacteria through the blood producing a peak in mice death between 24and 48 hours post-infection. Therefore, from the bacteria counts in theorgans and blood can be obtained a measure of the invasive andreplicative capability of a particular strain.

In FIG. 43 we observed a marked difference between the bacterial load inkidneys, spleen and blood from mice administered with Δmurl/Δdat straincompared to mice infected with wild type strain, over the time. Evenwhen a maximum peak of infection is expected between 24 and 48 hours, nocolonies were recovered for the Δmurl/Δdat strain on post-infection days1 nor 2. In fact, CFU recovery of virulent wild type strain reachedmaximum counts on these post-infection even thought an inoculum 1:13diluted was administered.

Consequently, “in vivo” clearance of Δmurl/Δdat strain occurs before 24hours which implies an appropriate security level for its administrationas live-attenuated vaccine.

Example 33 Passive Inmunization with S. aureus 132 Δmurl/Δdat VaccineAntisera

We next explored the efficacy of vaccine serum from mice immunized withthe S. aureus 132 Δmurl/Δdat strain to protect mice againststaphylococcal infection.

Vaccine or naive sera (150 μL) were injected into peritoneal cavity ofBALB/c mice (n=5) 3.5 h prior to challenge with a 5× dose of virulent S.aureus 132 wild type strain and survival of mice was monitored for 14days.

As shown in FIG. 53, all mice passively immunized with vaccine serumwere protected from challenge, whereas 60% of the mice receiving naïveserum succumbed to infection (P=0.0429; Mantel-Cox test (log-ranktest)).

These results demonstrate that the transfer of S. aureus antiserum,generated with the double mutant Δmurl/Δdat strain, confers significantlevel of protection of mice and prevents death when administered 3.5hours prior to infection with virulent S. aureus 132 wild type strain.

Example 34 Exploitation of Different routes for Vaccine Administration

Both, schedule and route of administration could determine the potentialimmunogenicity of a particular vaccine being key factors in the finalsuccess of a vaccination procedure.

Therefore, we evaluated if the antibody immune response (IgG) elicitedby using different routes of administration—intraperitoneal,intramuscular, subcutaneous and intranasal could be affected by thisvariable. Also, the administration schedule was considered, namely thenumber of doses and vaccine dosage (bacterial inocula content).

All routes have both advantages and disadvantages, such as theabsorption, bioavailability and metabolism of the substance. At present,the majority of human vaccines approved are parenteral, using theintramuscular, subcutaneous and intradermic routes for administration.Although intramuscular vaccination is considered till date as theultimate ways, nasal route offers easy of self administration, inductionof mucosal as well as systemic imunity. Also, both liquid and dry powerformulations can be given this way. Finally, intranasal administrationmay be best suited for barrier vaccinations, following the outbreak ofhighly infections diseases.

Mucosal membranes are intensively exposed to microorganisms and otherexternal agents so they are places of intense immune activity. A.baumannii, P. aeruginosa and S. aureus are common causes of respiratoryinfections (among many others) therefore the use of intranasalvaccination would be beneficial to prevent colonization and diseasecaused by these microorganisms.

When comparing the three parenteral routes tested we found nodifferences between intraperitoneal, subcutaneous and intramuscularadministration in the generation of antibody mediated immune response.Also, similar high antibody titers were found after intranasaladministration of A. baumanii- and P. aeruginosa-derived vaccines. Incontrast, S. aureus vaccine elicited IgG production in a lesser extentthrough mucosal is immunization (FIG. 48A-C). In general, repeatedimmunizations boosted IgG production over the time.

Considering vaccine dosages, different patterns of humoral response wereseen depending on the microorganism. Both tested doses of thelive-attenuated A. baumani- and P. aeruginosa-vaccines (1× and 0.1×;0.4× and 0.04×; respectively), administrated using parenteral routes(also intranasal route for P. aeruginosa), triggered the production ofantibodies efficiently (FIG. 48A-B). In the case of S. aureus-vaccine(FIG. 48C), a marked reduction in IgG titers were observed when thelowest dose (1×) was used to immunize mice by intranasal route.Nevertheless, the use of parental routes for vaccination was effectiveeven using the lowest bacterial dosages (0.2× for intraperitoneal andintramuscular routes; and 1× for subcutaneous).

In order to assess the efficacy of the different routes of vaccineadministration for mice protection, once immunization schedules werefinished with P. aeruginosa-derived vaccine, mice were challenged with alethal dose (0.4×) of the wild type PAO1 strain (administeredintraperitoneally as described previously) and survival was monitored asabove. As expected with the high antibody titers produced after the5^(th) administration of the vaccine, all mice survived demonstrating agood correlation between IgG and protection. Thus, all theadministration routes evaluated can be suitable for preventing acutesepsis caused by this microorganism.

SEQUENCE LISTING SEQ ID No 1: Gutamate racemase MurI ofA. baumannii ATCC 17978 (A3MIP5_ACIBT)MTAIQPLFTELEPMPKALADAPIGIFDSGIGGMSVAAEIAKYLPNERIVYYADTAYVPYGPRSDEEIRELTARAVDWLYRQGCKIAVVACNTASAFSLDHLREHYGEHFPIVGLVPALKPAVLQTRSKVVAVLATPATFRGQLIKDVVEKFAVPAGVKVMTLTSLELVPCVEAGQQMSPVCLNALREVLQPAVEQGADYLVLGCTHYPFLNEAIHHLFDNQFTLVDSGLAVARQTARILIKNELLCDQIRQNVARIECYVSGNNADALQPVLQNMIPQELTWTLHNLSSEQ ID No 2: Glutamate racemase MurI ofA. baumannii ATCC 17978 (A3MA43_ACIBT)MNNNNNPIGMIDSGLGGLSLFKYIRQALPNEDIIYFADSKYVPYGDRESDWIVSRTTHLISNLVTHGKCKAIVIACNTMTAVAVETIRAQINVPLIAIEPAVKPAVAMTLSKHIAVLATATTVKGKNLKSLIETYAQDIKVSLVPCIGLAEKIETGKAHTAEVKDYLKNILAPLVEQKVDTIILGCTHYPFVSDTIQEIVGRDIQIIEPSEAVTAQLIRQLNQYHLSSESPNEGNHIIWTSSDPLEVADV TFSLLGTRLPVETTDFSEQ ID No 3: Glutamate racemase MurI of Escherichia coli (MURI_ECOLI)MATKLQDGNTPCLAATPSEPRPTVLVFDSGVGGLSVYDEIRHLLPDLHYIYAFDNVAFPYGEKSEAFIVERVVAIVTAVQERYPLALAVVACNTASTVSLPALREKFDFPVVGVVPAIKPAARLTANGIVGLLATRGTVKRSYTHELIARFANECQIEMLGSAEMVELAEAKLHGEDVSLDALKRILRPWLRMKEPPDTVVLGCTHFPLLQEELLQVLPEGTRLVDSGAAIARRTAWLLEHEAPDAKSADANIAFCMAMTPGAEQLLPVLQRYGFETLEKLAVLGSEQ ID No 4: Glutamate racemase MurI ofPseudomonas aeruginosa (MURI_PSEAE)MAVESAAVGVFDSGVGGLSVLREIRARLPSESLLYVADNAHVPYGEKSAEYIRERCERIGDFLLEQGAKALVLACNTATAAAAAELRERYPQVPLVAMEPAVKPAAAATRNGRVGVLATTGTLKSARFAALLDRFASDVQVFTQPCPGLVERIEAGDLYGPQTRALLERLLAPILEQGCDTLILGCTHYPFVKPLLAELIPAEMAVIDTGAAVARQLERVLSARALLAS GQAATPRFWTSALPEEMERILPILWGSPESVGKLVVSEQ ID No 5: Glutamate racemase MurI of Staphylococcus aureus 132 (MURI)MNKPIGVIDSGVGGLTVAKEIMRQLPNETIYYLGDIGRCPYGPRPGEQVKQYTVEIARKLMEFDIKMLVIACNTATAVALEYLQKTLSIPVIGVIEPGARTAIMTTRNQNVLVLGTEGTIKSEAYRTHIKRINPHVEVHGVACPGFVPLVEQMRYSDPTITSIVIHQTLKRWRNSESDTVILGCTHYPLLYKPIYDYFGGKKTVISSGLETAREVSALLTFSNEHASYTEHPDHRFFATGDPTHITNIIK EWLNLSVNVERISVNDSEQ ID No 6: D-amino acid transaminase Datof Staphylococcus aureus 132 (DAT)MEKIFLNGEFVSPSEAKVSYNDRGYVFGDGIYEYIRVYNGKLFTVTEHYERFLRSANEIGLDLNYSVEELIELSRKLVDMNQIETGAIYIQATRGVAERNHSFPTPEVEPAIVAYTKSYDRPYDHLENGVNGVTVEDIRWLRCDIKSLNLLGNVLAKEYAVKYNAVEAIQHRGETVTEGSSSNAYAIKDGVIYTHPINNYILNGITRIVIKKIAEDYNIPFKEETFTVDFLKNADEVIVSSTSAEVTPVIKLDGEPVNDGKVGPITRQLQEGFEKYIESHSISEQ ID No 7: UP0380(NotI)F: CCCGCGGCCGCGGGGTCCTGCACCTACGATGASEQ ID No 8: UP0380(BamHI)R: CCCGGATCCGGGACCTCCAATACCTGAATCSEQ ID No 9: DOWN0380(BamHI)F: CCCGGATCCGGGGCTCTGTTGTAGGCATTCSEQ ID No 10: DOWN0380(SphI)R: CCCGCATGCGGGCATCCTTGTGATTGCATTSEQ ID No 11: UP3398(NotI)F_II: CCCGCGGCCGCGGGTTGGTCAGGTCCTTGTTGSEQ ID No 12: UP3398(BamHI)R_II: CCCGGATCCGGGTACAGCCGTCATGGTGTTSEQ ID No 13: DOWN3398(BamHI)F: CCCGGATCCGGGACGCGTTTACCTGTAGAASEQ ID No 14: DOWN3398(SphI)R: CCCGCATGCGGGAGCGGTACAACTAATTGGSEQ ID No 15: EXTFW0380: GCAATTAGGCACTTGAGG SEQ ID No 16: EXTRV0380:ATACGCTCAGGTTGCATC SEQ ID No 17: INTFW0380: AGCCTATGTTCCGTATGGSEQ ID No 18: INTRV0380: TCAACCAGTGTGAATTGG SEQ ID No 19: EXTFW3398:CCGATTGGAATGATTGAC SEQ ID No 20: EXTRV3398: AGAGCATTCTGGTCGAAGSEQ ID No 21: INTFW3398: TAGCAATAGAACCAGCGG SEQ ID No 22: INTRV3398:TTGTGCCGTTACAGCTTC SEQ ID No 23: UPPA4662(HindIII)F_II:CCCAAGCTTGGGGGCAATCCGCCGTATATC SEQ ID No 24: UPPA4662(NotI)R:CCCGCGGCCGCGGGGGCGTTGCCCGCAGACGG SEQ ID No 25: DOWNPA4662(NotI)F:CCCGCGGCCGCGGGTCGTTCCTGGCAGACGTG SEQ ID No 26: DOWNPA4662(XbaI)R:CCCTCTAGAGGGTCCGCTCTCGCAGTCCGA SEQ ID No 27: EXTFWPA4662:GTATCGGCAAGGTGGAGT SEQ ID No 28: EXTRVPA4662: GAATGGCTTGATCGAGTCSEQ ID No 29: INTFWPA4662: ATCCGAATCGTTGCTCTA SEQ ID No 30: INTRVPA4662:ACAATACGCGCTCCAGCT sEQ ID No 31: murIUP(MluI)F:CCCACGCGTGGGCCGAAACAAAAAACAGTA SEQ ID No 32: murIUP(NotI)R:CCCGCGGCCGCGGGATTCGGTCATCCTTACTT SEQ ID No 33: murIDOWN(NotI)F:CCCGCGGCCGCGGGGAGGATTTTTAATGAAAG SEQ ID No 34: murIDOWN(BglII)R:CCCAGATCTGGGTTTCTTCCATTGAACTTC SEQ ID No 35: murIF: TGTCGGAGGTTTGACAGTAGSEQ ID No 36: murIR: CTAACTTCACGAGCCGTTTC SEQ ID No 37: murIExtF:GCTTGCCCTAAAGGTATTCC SEQ ID No 38: murIExtR: GGGCCACTCATACTTATGACSEQ ID No 39: murIseqF: ATGACTGAACAATCAGTGAA SEQ ID No 40: murIseqR:TGATGGTGCCATGTAAAGTT SEQ ID No 41: datUP(MluI)F:CCCACGCGTGAAACGTATTCATATGAT SEQ ID No 42: datUP(NotI)R:CCCGCGGCCGCATATTATTCCTCCACGCA SEQ ID No 43: datDOWN(NotI)F:CCCGCGGCCGCAATTCTTTCATCATATTT SEQ ID No 44: datDOWN(BglII)R:CCCAGATCTGCGAATCTAAACTCGGTA SEQ ID No 45: datF: TATTCAAGCAACGCGTGGTGSEQ ID No 46: datR: AGTTGACGTGTAATTGGGCC SEQ ID No 47: datExtF:GTCATGGGTGACGTGACAAC SEQ ID No 48: datExtR: GCACCACCTGCTGAATCAAGSEQ ID No 49: datseqF: GCCGGTTGTAACAGAAGATG SEQ ID No 50: datseqR:CAATTGCCGGGTCTGCAATC

1. A method for the production of a pharmaceutical composition,preferably a vaccine, comprising mutant live auxotrophic bacterialstrains for D-glutamate, wherein the pharmaceutical composition issuitable for the prophylactic treatment (before infection) and/ortherapeutic treatment (after infection or after the clinicalmanifestation of the disease caused by the infection) of animals and/orhumans against infection with the wild type form of the mutantauxotrophic bacterial strain of the composition, and wherein saidpharmaceutical composition is produced by a method comprising the stepsof: a. obtaining mutant live auxotrophic bacterial strains forD-glutamate; b. introducing said mutant live auxotrophic bacerialstrains in a pharmaceutically acceptable carrier or diluent andoptionally adding an adjuvant; and c. Optionally freeze-drying thepharmaceutical composition.
 2. A method for the production of apharmaceutical composition, preferably a vaccine, comprising mutant liveauxotrophic bacterial strains for D-glutamate, wherein thepharmaceutical composition is suitable for the prophylactic (beforeinfection) and/or therapeutic treatment (after infection) of animalsand/or humans against infection with the wild type form of the mutantauxotrophic bacterial strain of the composition, and wherein saidpharmaceutical composition is produced by a method comprising the stepsof: a. providing a bacterial strain capable of expressing glutamateracemase and possibly D-amino acid transaminase and comprising apeptidoglycan cell wall; b. inactivating the gene or genes encoding forthe glutamate racemase enzyme and, if needed, the gene or genes encodingfor the enzyme D-amino acid transaminase in such way that the bacterialstrain is no longer capable of expressing a functional glutamateracemase and/or a functional D-amino acid transaminase, wherein theinactivation of said genes thus causes said bacterial strain to beauxotrophic for D-glutamate; and c. introducing said mutant liveauxotrophic bacterial strains in a pharmaceutically acceptable carrieror diluent and optionally adding an adjuvant; and d. Optionallyfreeze-drying the pharmaceutical composition.
 3. The method for theproduction of a pharmaceutical composition of claim 1 or 2, wherein thepharmaceutical composition is a vaccine and the production methodcomprises adding an adjuvant. 4, The method of any of claims 1-3,wherein the bacterial strain of step a) is a gram positive or gramnegative bacteria.
 5. The method of claim 2, wherein the bacterialstrain of step a) has as the only way of synthesis of D-glutamate theglutamate racemase enzyme and wherein step b) of the method comprisesthe inactivation of the genes encoding for this enzyme, namely forglutamate racemase.
 6. The method of any of claims 1-3, wherein thebacterial strain of step a) is selected from the list of bacterialspecies consisting of: Acinetobacter baumannii, Acinetobacter baylyi,Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacterjunii, Acinetobacter lwoffii, Acinetobacter nosocomialis, Acinetobacterpittii, Acinetobacter radioresistens, Actinobacillus lignieresii,Actinobacillus suis, Aeromonas caviae, Aeromonas hydrophila, Aeromonasveronii subsp. sobria, Aggregatibacter actinomycetemcomitans, Arcobacterbutzleri, Arcobacter nitrofigilis, Bacillus amyloliquefaciens, Bacillusanthracis, Bacillus bataviensis, Bacillus cellulosilyticus, Bacilluscereus, Bacillus clausil, Bacillus licheniformis, Bacillus megaterium,Bacillus pumilus, Bacillus subtilis, Bacillus thuringiensis, Bacteroidesfragilis, Bordetella avium, Bordetella bronchiseptica, Bordetellapertusis, Bordetella petrii, Brucella abortus, Brucella melitensis,Brucella suis, Burkholderia cenocepacia, Burkholderia mallei,Burkholderia multivorans, Burkholderia pseudomallei, Burkholderiathailandensis, Campylobacter concisus, Campylobacter fetus subsp. fetus,Campylobacter fetus subsp. venerealis, Campylobacter gracilis,Campylobacter hominis, Campylobacter jejuni, Campylobacter rectus,Campylobacter showae, Campylobacter upsaliensis, Citrobacter freundii,Citrobacter koseri, Clostridium asparagiforme, Clostridium botulinum,Clostridium butyricum, Clostridium difficile, Clostridium perfringens,Clostridium saccharobutylicum, Clostridium tetani, Corynebacteriumdiphtheriae, Corynebacterium pseudotuberculosis, Enterobacter aerogenes,Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium,Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacteriumnecrophorum, Fusobacterium nucleatum, Granulicatella adiacens,Granulicatella elegans, Haemophilus equigenitalis, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus paragaffinarum,Haemophilus parasuis, Haemophilus pleuropneumoniae, Haemophilus somnus,Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae,Legionella oakridgensis, Legionella pneumophila, Leptospira biflexa,Leptospira illini, Leptospira interrogans, Listeria monocytogenes,Lysinibacillus fusiformis, Lysinibacillus sphaericus, Moraxella bovis,Morganella morganii, Mycobacterium abscessus, Mycobacterium africanum,Mycobacterium avium, Mycobacterium bovis, Mycobacterium leprae,Mycobacterium tuberculosis, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Propionibacterium acnes, Proteus hanseri, Proteus mirabilis, Pseudomonasaeruginosa, Salmonella cholerasuis, Salmonella enterica subsp. enterica,Salmonella enteritidis, Salmonella paratyphi, Salmonella typhi, Serratiaplymuthica, Shigella boydii, Shigella dysenteriae, Shigella flexneri,Staphylococcus arlettae, Staphylococcus aureus, Staphylococcus capitis,Staphylococcus caprae, Staphylococcus carnosus, Staphylococcusepidermidis, Staphylococcus equorum, Staphylococcus haemolyticus,Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcuspasteuri, Staphylococcus pettenkoferi, Staphylococcus pseudointermedius,Staphylococcus saprophyticus, Staphylococcus simiae, Staphylococcussimulans, Staphylococcus warneri, Stenotrophomonas maltophilia,Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcusdysgalactiae subsp. equisimilis, Streptococcus equi, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis..
 7. The method of claim 6,wherein said bacterial strain of step a) is selected from the listconsisting of the following species: Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus.
 8. The method of claim7, wherein said bacterial strain of step a) is the bacterial strain ofA. baumannii designated Acinetobacter baumannii Delta0380/Delta3398 anddeposited under the Budapest treaty before the Spanish Type CultureCollection on Apr. 14, 2014 with strain number 8588 by FundaciónProfesor Novoa Santos.
 9. The method of claim 7 wherein said bacterialstrain of step a) is the bacterial strain of P. aeruginosa designatedPseudomonas aeruginosa DeltaPA4662 and deposited under the Budapesttreaty before the Spanish Type Culture Collection on Apr. 14, 2014 withstrain number 8589 by Fundación Profesor Novoa Santos.
 10. The method ofclaim 7 wherein said bacterial strain of step a) is the bacterial strainof S. aureus designated 132deltamurl/deltadat and deposited under theBudapest treaty before the Spanish Type Culture Collection on Jun. 11,2014 with strain number 8587 by Fundación Profesor Novoa Santos.
 11. Apharmaceutical composition, preferably a vaccine, comprising mutant liveauxotrophic bacerial strains for D-glutamate and a pharmaceuticallyacceptable carrier or diluent and optionally an adjuvant, wherein saidpharmaceutical composition is suitable for the prophylactic (beforeinfection) and/or therapeutic treatment (after infection or after theclinical manifestation of the disease caused by the infection) ofanimals and/or humans against infection with the wild type form of themutant auxotrophic bacterial strain of the composition.
 12. Thepharmaceutical composition of claim 11, wherein said pharmaceuticalcomposition is a vaccine and wherein optionally said vaccine comprisesan adjuvant.
 13. The pharmaceutical composition of any of claims 11 to12, wherein said pharmaceutically acceptable carrier or diluent isselected from the list consisting of water, culture fluid, a solution ofphysiological salt concentration and/or stabilisers such as SPGA,carbohydrates (e.g. sorbitol, mannitol, starch, sucrose, glucose,dextran), proteins such as albumin or casein, protein containing agentssuch as bovine serum or skimmed milk and buffers (e.g. phosphatebuffer).
 14. The pharmaceutical composition of any of claims 11 to 13,wherein the adjuvant is selected from the list consisting of FreundsComplete and Incomplete adjuvant, vitamin E, non-ionic block polymers,muramyldipeptides, ISCOMs (immune stimulating complexes), Saponins,mineral oil, vegetable oil, Carbopol, the E. coli heat-labile toxin (LT)or Cholera toxin (CT), aluminium hydroxide, aluminium phosphate oraluminium oxide, oil-emulsions (e.g. of Bayol F® or Marcol 52®, saponinsand vitamin-E solubilisate.
 15. The pharmaceutical composition of any ofclaims 11 to 14, wherein said pharmaceutical composition comprises adose of mutant live auxotrophic bacterial strains for D-glutamateranging between 10³ and 10¹⁰ bacteria.
 16. The pharmaceuticalcomposition of any of claims 11 to 15, wherein said pharmaceuticalcomposition is in a freeze-dried form.
 17. The pharmaceuticalcomposition of any of claims 11 to 16, wherein the bacterial strain isselected from the list of bacterial species consisting of: Acinetobacterbaumannii, Acinetobacter baylyi, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter lwoffii,Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacterradioresistens, Actinobacillus lignieresii, Actinobacillus suis,Aeromonas caviae, Aeromonas hydrophila, Aeromonas veronii subsp. sobria,Aggregatibacter actinomycetemcomitans, Arcobacter butzleri, Arcobacternitrofigilis, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillusbataviensis, Bacillus cellulosilyticus, Bacillus cereus, Bacillusclausii, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus subtilis, Bacillus thuringiensis, Bacteroides fragilis,Bordetella avium, Bordetella bronchiseptica, Bordetella pertusis,Bordetella petrii, Brucella abortus, Brucella melitensis, Brucella suis,Burkholderia cenocepacia, Burkholderia mallei, Burkholderia multivorans,Burkholderia pseudomallei, Burkholderia thailandensis, Campylobacterconcisus, Campylobacter fetus subsp. fetus, Campylobacter fetus subsp.venerealis, Campylobacter gracilis, Campylobacter hominis, Campylobacterjejuni, Campylobacter rectus, Campylobacter showae, Campylobacterupsaliensis, Citrobacter freundii, Citrobacter koseri, Clostridiumasparagiforme, Clostridium botulinum, Clostridium butyricum, Clostridiumdifficile, Clostridium perfringens, Clostridium saccharobutylicum,Clostridium tetani, Corynebacterium diphtheriae, Corynebacteriumpseudotuberculosis, Enterobacter aerogenes, Enterobacter cloacae,Enterococcus faecalis, Enterococcus faecium, Erysipelothrixrhusiopathiae, Escherichia coli, Fusobacterium necrophorum,Fusobacterium nucleatum, Granulicatella adiacens, Granulicatellaelegans, Haemophilus equigenitalis, Haemophilus influenzae, Haemophilusparainfluenzae, Haemophilus paragallinarum, Haemophilus parasuis,Haemophilus pleuropneumoniae, Haemophilus somnus, Helicobacter pylon,Klebsiella oxytoca, Klebsiella pneumoniae, Legionella oakridgensis,Legionella pneumophila, Leptospira biflexa, Leptospira Leptospirainterrogans, Listeria monocytogenes, Lysinibacillus fusiformis,Lysinibacillus sphaericus, Moraxella bovis, Morganella morganii,Mycobacterium abscesses, Mycobacterium africanum, Mycobacterium avium,Mycobacterium bovis, Mycobacterium leprae, Mycobacterium tuberculosis,Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida,Plesiomonas shigelloides, Propionibacterium acnes, Proteus hanseri,Proteus mirabilis, Pseudomonas aeruginosa, Salmonella cholerasuis,Salmonella enterica subsp. enterica, Salmonella enteritidis, Salmonellaparatyphi, Salmonella typhi, Serratia plymuthica, Shigella boydii,Shigella dysenteriae, Shigella flexneri, Staphylococcus arlettae,Staphylococcus aureus, Staphylococcus capitis, Staphylococcus caprae,Staphylococcus carnosus, Staphylococcus epidermidis, Staphylococcusequorum, Staphylococcus haemolyticus, Staphylococcus hominis,Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcuspettenkoferi, Staphylococcus pseudointermedius, Staphylococcussaprophyticus, Staphylococcus simiae, Staphylococcus simulans,Staphylococcus warneri, Stenotrophomonas maltophilia, Streptococcusagalactiae, Streptococcus dysgalactiae, Streptococcus dysgalactiaesubsp. equisimilis, Streptococcus equi, Streptococcus pneumoniae,Streptococcus pyogenes, Streptococcus uberis, Streptococcuszooepidermicus, Taylorella asinigenitalis, Taylorella equigenitalis,Treponema carateum, Treponema cuniculi, Treponema hyodisenteriae,Treponema pallidum, Treponema suis, Veillonella atypica, Veillonelladispar, Veillonella parvula, Veillonella ratti, Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificans, Yersinia enterocolitica, Yersiniapestis and Yersinia pseudotuberculosis.
 18. The pharmaceuticalcomposition of claim 17, wherein the bacterial strain is selected fromthe list consisting of the following species: Acinetobacter baumannii,Pseudomonas aeruginosa and Staphylococcus aureus.
 19. The pharmaceuticalcomposition of claim 18, wherein the bacterial strain is the bacterialstrain of A. baumannii designated Acinetobacter baumanniiDelta0380/Delta3398 and deposited under the Budapest treaty before theSpanish Type Culture Collection on Apr. 14, 2014 with strain number 8588by Fundación Profesor Novoa Santos.
 20. The pharmaceutical compositionof claim 18, wherein the bacterial strain is is the bacterial strain ofP. aeruginosa designated Pseudomonas aeruginosa DeltaPA4662 anddeposited under the Budapest treaty before the Spanish Type CultureCollection on Apr. 14, 2014 with strain number 8589 by FundaciónProfesor Novoa Santos.
 21. The pharmaceutical composition of claim 18,wherein the bacterial strain is is the bacterial strain of S. aureusdesignated . . . and deposited under the Budapest treaty before theSpanish Type Culture Collection on Apr. 14, 2014 with strain number . .. by Fundación Profesor Novoa Santos.
 22. A mutant live auxotrophicbacterial strain for D-glutamate, wherein said bacterial strain is thebacterial strain of P. aeruginosa designated Pseudomonas aeruginosaDeltaPA4662 and deposited under the Budapest treaty before the SpanishType Culture Collection on Apr. 14, 2014 with strain number 8589 byFundación Profesor Novoa Santos.
 23. A mutant live auxotrophic bacterialstrain for D-glutamate, wherein said bacterial strain is the bacterialstrain of S. aureus designated 132deltamurl/deltadat and deposited underthe Budapest treaty before the Spanish Type Culture Collection on Jun.11, 2014 with strain number 8587 by Fundación Profesor Novoa Santos. 24.The bacterial strain as defined in any of claim 22 or 23, for use as amedicament, in particular as a vaccine.
 25. The pharmaceuticalcomposition of any of claims 1 to 21 or the mutant live auxotrophicbacterial strain for D-glutamate of any of claim 22 or 23, for use in amethod of prophylactic treatment (before infection) and/or therapeutictreatment (after infection or after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacterialstrain of the composition.
 26. An antibody or fragment thereof selectedfrom the group consisting of Fab, F(ab′)2, Fv, scFv, di-scFv and sdAB,capable of recognizing a mutant live auxotrophic bacterial strain forD-glutamate, wherein said antibody or fragment thereof is suitable forthe prophylactic treatment (before infection) and/or therapeutictreatment (after infection or after the clinical manifestation of thedisease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacterialstrain of the composition.
 27. An antibody or fragment thereof selectedfrom the group consisting of Fab, F(ab′)2, Fv, scFv, di-scFv and sdAB,obtained or obtainable after immunization of a mammal with a mutant liveauxotrophic bacterial strain for D-glutamate, wherein said antibody orfragment thereof is suitable for the prophylactic treatment (beforeinfection) and/or therapeutic treatment (after infection or after theclinical manifestation of the disease caused by the infection) ofanimals and/or humans against infection with the wild type form of themutant auxotrophic bacterial strain of the composition
 28. Apharmaceutical composition, preferably a vaccine, comprising theantibodies or fragments thereof of any of claim 26 or 27 and apharmaceutically acceptable carrier or diluent and optionally anadjuvant, wherein said pharmaceutical composition is suitable for theprophylactic (before infection) and/or therapeutic treatment (afterinfection or after the clinical manifestation of the disease caused bythe infection) of animals and/or humans against infection with the wildtype form of the mutant auxotrophic bacterial strain of the composition.29. The pharmaceutical composition of claim 28, wherein saidpharmaceutical composition is a vaccine and wherein optionally saidvaccine comprises an adjuvant.
 30. The antibodies or fragments thereofof any of claim 26 or 27, for use in therapy, in particular for use inpassive immunization.
 31. The pharmaceutical composition of claim 28 orthe antibodies or fragments thereof of any of claim 26 or 27, for use ina method of prophylactic treatment (before infection) and/or therapeutictreatment (after infection or after after the clinical manifestation ofthe disease caused by the infection) of animals and/or humans againstinfection with the wild type form of the mutant auxotrophic bacterialstrain of the composition.
 32. The pharmaceutical composition of any ofclaims 11-21 or claim 28 or the mutant live auxotrophic bacterial strainfor D-glutamate of any of claim 22 or 23 or the antibodies or fragmentsthereof of any of claim 26 or 27, for use in a method of prophylactictreatment (before infection) and/or therapeutic treatment (afterinfection or after after the clinical manifestation of the diseasecaused by the infection) of animals and/or humans against infection withthe wild type form of the mutant auxotrophic bacterial strain of thecomposition and wherein said composition, bacterial strain or antibodyor fragment thereof is administered intranasally, intradermally,subcutaneously, orally, by aerosol, intramuscularly, wing web andeye-drop administration.
 33. A kit or device comprising the antibody orfragment thereof of any of claim 26 or
 27. 34. A kit or device fordetecting an infection of bacterial origin through an immunoassaycomprising: (i) a first antibody called “capture antibody” as defined inany of claim 26 or 27, wherein said first antibody is preferablyattached to a solid support; (ii) a second labeled antibody called“detection antibody” which recognizes a region other than the regionrecognized by the first antibody , wherein said second antibodycomprises a marker which may be fluorescent , luminescent or an enzyme;(iii) a reagent showing affinity for the second antibody, said reagentbeing coupled to a first member of a binding pair; and (iv) a secondmember of a binding pair coupled to a fluorescent, luminescent or anenzyme, wherein the binding pair is selected from the group consistingof: hapten and antibody; antigen and antibody; biotin and avidin; biotinand streptavidin; a biotin analogue and avidin; a biotin analogue andstreptavidin; sugar and lectin; an enzyme and a cofactor; a nucleic acidor a nucleic acid analogue and the complementary nucleic acid or nucleicacid analogue.
 35. Use of the kit or device of any of claim 33 or 34 forthe qualitative and/or quantitative determination of bacterial speciesor bacterial strains in a biological sample from a mammal, inparticular, in the plasma of a mammal suspected of suffering from abacterial disease.
 36. A method of in vitro cultivation of bacterialstrains auxotrophic for D-glutamate comprising the utilization of aconcentration of D-glutamate between 0.00001 and 120 mM.
 37. The methodof claim 36, wherein the concentration range of D-glutamate is between0.01-50 mM.
 38. The method of claim 37, wherein the concentration rangeof D-glutamate is between 10-20 mM.